U.S. patent number 4,201,770 [Application Number 05/936,876] was granted by the patent office on 1980-05-06 for antigenic modification of polypeptides.
This patent grant is currently assigned to The Ohio State University. Invention is credited to Vernon C. Stevens.
United States Patent |
4,201,770 |
Stevens |
May 6, 1980 |
Antigenic modification of polypeptides
Abstract
Modified hormones or fragments of hormones are useful in
producing antibodies when administered to an animal. Said
antibodies in turn cause neutralization of endogenous natural
protein hormones. The modification may be accomplished by attaching
various kinds of modifying groups to the hormone or fragment.
Modification may, for example, be achieved by chemically coupling
diazosulfanilic acid groups to the hormone or fragment. The protein
hormones to which this procedure can be applied are mammalian
protein reproductive hormones such as, for example, Follicle
Stimulating Hormone (FSH), or Human Chorionic Gonadotropin (HGG).
These modified hormone or fragment may be administered to animals
for the purpose of contraception, abortion, or treatment of hormone
related disease states and disorders.
Inventors: |
Stevens; Vernon C. (Dublin,
OH) |
Assignee: |
The Ohio State University
(Columbus, OH)
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Family
ID: |
26999835 |
Appl.
No.: |
05/936,876 |
Filed: |
August 25, 1978 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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622031 |
Oct 14, 1975 |
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462955 |
Apr 22, 1974 |
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406821 |
Oct 16, 1973 |
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357892 |
May 7, 1973 |
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Current U.S.
Class: |
424/185.1;
514/9.9; 514/21.5; 514/10.1; 424/195.11; 514/841; 424/198.1;
424/194.1; 514/1; 530/324 |
Current CPC
Class: |
A61K
39/0006 (20130101); C07K 14/59 (20130101); A61K
38/00 (20130101); A61K 2039/6012 (20130101); A61K
2039/5555 (20130101); Y10S 514/841 (20130101) |
Current International
Class: |
A61K
39/00 (20060101); C07K 14/435 (20060101); C07K
14/59 (20060101); A61K 38/00 (20060101); A61K
037/00 (); C07C 103/52 (); A61K 039/00 () |
Field of
Search: |
;260/112.5R
;424/88,177 |
Other References
Tung, Int. J. Fertil. 21, 197-206, 1976. .
Pilsworth, New Scientist, 1978, pp. 665-667. .
Laurence, et al., Internat. Journal of Fertility, 1969, 14, 8-15.
.
Laurence, et al., Endocrinology, 82, 1190-1199, 1968. .
Immunochemistry, 1968, 5, 55-65. .
Brit. Med., Bull., 26, 72-78, 1970. .
Endocrinology 87, 1181-1185, 1970. .
Proc. Soc. Expte. Biol. and Med., 133 (1920), 410-415..
|
Primary Examiner: Phillips; Delbert R.
Attorney, Agent or Firm: Millard, Cox & Smith
Parent Case Text
This application is a continuation-in-part of Ser. No. 622,031
filed Oct. 14, 1975, now abandoned which in turn is a
continuation-in-part of Ser. No. 462,955 filed Apr. 22, 1974, which
in turn is a continuation-in-part of Ser. No. 406,821, filed Oct.
16, 1973, which in turn is a continuation-in-part of Ser. No.
357,892, filed May 7, 1973 now abandoned.
Claims
What is claimed is:
1. A method for controlling fertility in primate animals having
naturally occurring endogenous Chroionic Gonadotropin hormone by
neutralizing the biological action of the endogenous hormone,
comprising the steps of:
administering to said primate animal an immunologically effective
amount of the hormone, a subunit thereof, a peptide fragment of the
subunit or a synthetically derived peptide having a sequence
analogous to at least a portion of said subunit;
said hormone, subunit, fragment or synthetically derived peptide
being modified by the coupling thereof with a non-endogenous
material to effect the formation, following said administration, of
antibodies having a specificity to endogenous Chorionic
Gonadotropin;
thereby inhibiting the fertility of said primate animal by
preventing one or more normal biological functions of the
endogenous Chorionic Gonadotropin hormone.
2. The method of claim 1 wherein said modified hormone, subunit,
fragment or synthetically derived peptide is administered in
combination with an adjuvant.
3. The method of controlling fertility in primate animals having
naturally occuring endogenous Chorionic Gonadotropin hormone
comprising the steps of:
providing a quantity of the hormone, a subunit thereof, a peptide
fragment thereof, or a synthetically derived peptide having a
sequence analogous to at least a portion of said subunit and being
non-antigenic within said primate animals;
modifying said hormone, subunit, fragment, or synthetically derived
peptide by the coupling thereof with a non-endogenous material;
administering to said primate animal an immunologically effective
amount of said modified hormone, subunit, fragment, or
synthetically derived peptide;
thereby inhibiting the fertility of said primate animals by
preventing one or more normal biological functions of endogenous
Chorionic Gonadotropin.
4. The method of claim 3 wherein said modified hormone, subunit,
fragment or synthetically derived peptide is administered in
combination with an adjuvant.
5. The method of claim 3 wherein said non-endogenous material is
coupled with said hormone, subunit, fragment or synthetically
derived peptide through a sulfhydryl-group linkage.
6. The method of claim 3 wherein said hormone, subunit, fragment or
synthetically derived peptide is one having a sulfhydryl group
therewithin, and said coupling is carried out by the treatment
thereof with an activator of the structure ##STR10## wherein X
represents a non-reacting connective entity so as to effect
reaction of the maleiimide group of the activator with a said
sulfhydryl group; and
treating the resultant activated hormone, subunit, fragment or
synthetically derived peptide with said non-endogenous
material.
7. The method of claim 6 wherein said non-reacting connective
entity, X, comprises an amino acid chain.
8. The method of claim 7 wherein said amino acid chain is a Prolene
spacer chain.
9. The method of claim 3 wherein said non-endogenous material
comprises sucrose copolymerized with epichlorohydrin.
10. The method of claim 3 wherein said non-endogenous material
comprises (poly)tyrosine, (poly)alanine, (poly)dextran,
thyroglobulin, or a combination thereof.
11. The method of claim 6 wherein said non-endogenous material
comprises the protein, flagellin.
12. The method of claim 6 wherein said non-endogenous material
material comprises an influenza virus.
13. The method of claim 12 in which said influenza virus is an
influenza subunit incorporating substantially only the viral
proteins Haemagglutin and Neuraminease.
14. The method of claim 6 wherein said non-endogenous material is
diptheria toxiod.
15. The method of claim 6 wherein said non-endogenous material is a
cholera organism.
16. The method of claim 6 wherein said activated hormone, subunit,
fragment or synthetically derived peptide is treated with said
non-endogenous material at slightly alkaline pH.
17. The method of claim 3 or 6 wherein said fragment which is
modified is of the chemical configuration:
Thr-Cys-Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Ser-Ser-Ser-Lys-Ala-Pro-Pro-Pro-Ser
-Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pro-Gly-Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln;
Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Pro-Pro-Pro-Cys-Pro-Pro-Pro-Ser-Asp-Thr-Pro
-Ile-Leu-Pro-Gln;
Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Pro-Pro-Pro-Pro-Pro-Pro-Cys-Pro-Pro-Pro-Pro
-Pro-Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln;
Asp-His-Pro-Leu-Thr-Aba-Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Ser-Ser-Ser-Lys-Als
-Pro-Pro-Pro-Ser-Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pro-Gly-Pro-Ser-Asp-Thr-Pro-Il
e-Leu-Pro-Gln-Pro-Pro-Pro-Pro-Pro-Pro-Cys;
Asp-His-Pro-Leu-Thr-Aba-Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Ser-Ser-Ser-Lys-Ala
-Pro-Pro-Pro-Ser-Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pro-Gly-Pro-Ser-Asp-Thr-Pro-Il
e-Leu-Pro-Gln-Cys;
or
Cys-Pro-Pro-Pro-Pro-Pro-Pro-Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Ser-Ser-Ser-Lys
-Ala-Pro-Pro-Pro-Ser-Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pro-Gly-Pro-Ser-Asp-Thr-Pr
o-Ile-Leu-Pro-Gln.
18. The method of claim 3 wherein said fragment which is modified
is of the chemical configuration:
Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Ser-Ser-Ser-Lys-Ala-Pro-Pro-Pro-Ser-Leu-Pro
-Ser-Pro-Ser-Arg-Leu-Pro-Gly-Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln;
Gln-Asp-Ser-Ser-Ser-Ser-Lys-Ala-Pro-Pro-Pro-Ser-Leu-Pro-Ser-Pro-Ser-Arg-Leu
-Pro-Gly-Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln,
Phe-Gln-Asp-Ser-Ser-Ser-Ser-Lys-Ala-Pro-Pro-Pro-Ser-Leu-Pro-Ser-Pro-Ser-Arg
-Leu-Pro-Gly-Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln;
or
Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Ser-Ser-Ser-Lys-Als-Pro-Pro-Pro-Ser-Leu-Pro
-Ser.
19. The method of claim 3 wherein said non-endogenous material is
one having an amino group and said coupling is carried out by
activating said non-endogenous material with an activator of the
structure: ##STR11## where X represents a non-reacting connective
entity so as to effect reaction of the activator with said amino
group; and
treating the activated non-endogenous material with said hormone,
subunit, fragment or synthetically derived peptide which has a
sulfhydryl group.
20. The method of claim 19 wherein said activation is carried out
under neutral or acid conditions.
21. The method of claim 3 wherein said modification is carried out
to effect the constitution of two or more immunological
determinants effective to elicit antibody response to the
endogenous hormone, Chorionic Gonadotropin.
22. The method of claim 3 wherein:
said fragment or synthetically derived peptide which is modified is
of the chemical configuration:
Cys-Pro-Pro-Pro-Pro-Pro-Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln
and said administration thereof is in combination with another,
different modified Chorionic Gonadotropin hormine, subunit thereof,
peptide fragment of the said subunit, or a synthetically derived
peptide having a sequence at least analogous to at least a portion
of the fragment, the said modification of which effects the
constitution of at least one immunological determinant.
23. The method of claim 3 wherein:
said fragment or synthetically derived peptide which is modified is
of the chemical configuration:
Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Pro-Pro-Pro-Pro-Pro-Pro-Cys
and said administration thereof is in combination with another,
different modified Chorionic Gonadotropin hormine, subunit thereof,
peptide fragment of the said subunit, or a synthetically derived
peptide having a sequence at least analogous to at least a portion
of the fragment, the said modification of which effects the
constitution of at least one immunological determinant.
24. The method of claim 1 wherein said animal is human and said
hormone is human Chorionic Gonadoptropin.
Description
BACKGROUND OF THE INVENTION
It is well known that antibodies are generated in humans and in
other animals in response to the presence of foreign antigens. It
is also known to confer immunity on an animal by administering an
antibody formed elsewhere. For instance, the patents to Michaelson
(U.S. No. 3,553,317), Friedheim (U.S. No. 2,388,260), Reusser (U.S.
No. 3,317,400) and Peterson (U.S. No. 3,376,198) relate to
production of antibodies, which when injected into an animal of a
different species or into a human being cause passive immunization.
In patents to Fell (U.S. No. 2,301,532 and U.S. No. 2,372,066), the
patentee refers to active immunization using modified histamine in
such animals as horses, cows, etc. In a paper by R. G. Edwards in
the British Medical Journal, Vol. 26, pages 72 to 78, published in
1970 on "Immunology of Conception and Pregnancy", he surveys the
literature regarding the possibilities of utilizing immunological
methods to influence or control fertility, surveying first
production of antibodies against tests or spermatozoa. Much of the
literature surveyed is directed to the production of foreign
antibodies which are injected into the subject (passive
immunization).
Hormone antibodies have been studied for a long time and the effect
of specific antisera have been recorded for many years. It is known
that administration of certain antibodies during pregnancy can
suppress implantation or cause fetal resorption. Several different
approaches have been tried ranging from the induction of near
permanent infertility in the case of agglutination of spermatozoa
in the male to the disturbance of a single pregnancy by passive
immunization with antibodies.
There are serious limitations to the use of passive immunization
procedures for human therapy. Since the antibodies are practically
produced only in non-human animals, the repeated injection of
animal proteins into humans is known to produce serious reaction in
many individuals.
British Patent Specification No. 1,058,828 discloses that small
molecules, referred to as "serological determinant peptides", can
be coupled to large protein molecules, such as cattle albumin and
the resultant conjugate then may be injected into animals for
antibody production. The document lists proteins from which the
serologically determinant peptides may be isolated prior to being
used in the process taught, the collection including viruses and
bacteria whose surface component has the characteristics of a
protein, toxins and hormones having protein structure and enzymes.
No specific hormone is named in the document and no utility of
anti-hormone immunization is described. The patent specification
references a publication entitled: "The Specificity of Serological
Reactions", Dover Publications, Inc., New York, 1962, Chapter V,
"Artificial Conjugated Antigens" by K. Landsteiner. This
publication outlines various chemical methods and applies them
passively to bind various toxic substances in the blood such as
arsenic. Thyroxine data provided in the publication suggests that
such methods may be applied to protein hormones without indicating
the therapeutic application, the publication teaching that specific
antibodies may be formed to the small molecules and these
antibodies are capable of neutralizing the biological action of a
large protein from which the small peptide was a part.
Recently it has been discovered that doses of certain steroids
consisting of synthetic non-protein hormones ("The Pill") when
administered at stated intervals usually confer protection against
pregnancy for a short time (possibly a month). This medication has
sometimes been found to create undesirable side effects in creating
undesirable metabolic changes and sometimes changes in the blood
clotting mechanisms. Moreover, the effect of each dose is of such
short duration that often it is of limited application,
particularly in remote areas to persons not readily instructed on
proper and continuing use.
There is need therefore of an effective safe method of creating a
temporary but relatively long-time immunity against pregnancy which
does not have serious side effects. There is also a need for an
effective safe method of terminating a pregnancy soon after
conception which does not have serious harmful side effects. Such
need may be met by the neutralization of a reproductive protein
which is necessary for the normal events of conception and/or
gestation.
There is also a need for a means for control of various disease
states or maladies caused or influenced by unusual excesses of
certain polypeptides such as gastrin, angiotension II, or
somatomedian. It is believed that this invention meets this need
safely and effectively.
SUMMARY OF THE INVENTION
This invention is concerned (1) with the production of antigens for
the purpose of active immunization, (2) with the antigens so
produced, and (3) with the use of said antigens. More particularly,
the invention relates to antigens consisting of natural protein
reproductive hormones, non-hormonal proteins, specific fragments of
such hormones and proteins and synthetically derived portions of
said hormones and proteins, all modified as will be indicated more
fully hereinafter. For the sake of simplicity, hereinafter in this
specification and in the claims, these antigens are collectively
referred to as modified polypeptides.
The invention is directed in one aspect to the use of modified
polypeptides in actively immunizing an animal, particularly
mammals, against the biological action of endogenous unmodified
non-hormonal natural protein and/or hormone. The state of immunity
arises because of the creation of antibodies which act against both
the antigenic modified polypeptide and its endogenous counterpart
which is neutralized (rendered biologically ineffectual) as a
result of the existence of said antibodies. The immunity may take
place because of the inability of the antibody to distinguish
between the modified polypeptide and the naturally existing
protein, but it is uncertain that this is in fact the situation. In
effect, the invention provides, in one aspect, for the
isoimmunization of a primate animal.
A more specific aspect of this invention relates to the
modification of protein reproductive hormones by adding certain
numbers of foreign moieties to each hormone molecule, or hormonal
fragment. The modification must be sufficient to cause the body to
create antibodies to the modified hormones which will neutralize or
inhibit the biological action of the natural hormones produced by
the body. Thus, the modified hormones become antigenic and cause
the production of antibodies which disrupt the natural processes of
conception and/or gestation. The term "protein reproductive
hormones" includes those hormones essential to the normal events of
the reproductive process.
According to a further aspect of this invention, a disease state
which can be treated by application of the technique of the instant
invention is the digestive disorder known to those skilled in the
medical field as the Zollinger-Ellison Syndrome. This syndrome or
disease state is generally described as a condition in which a
hyper secretion of the polypeptide gastrin, which is produced in
the pancreas and brings about a state of hyperacidity in the
stomach which results in a chronic digestive disorder. Heretofore,
the only effective treatment for this disease state was the
surgical removal of a part or total removal of the subject's
stomach. Although survival of such patients is usually not
threatened, the medical state and life style of such individuals is
severely affected by such treatment.
Treatment of such subjects with hapten coupled (produced according
to the general method described herein) or otherwise chemically
modified gastrin can be used to enhance the production of
antibodies against the hypersecretion of gastrin and thereby
alleviate or reduce the symptoms of this disease without surgical
intervention. Sufficient reduction by immunological means of this
substance in the system of the body would be sufficient to avoid
the complicated and serious consequences of the surgical treatment
currently in use. In practice, an effective amount of modified
gastrin is simply injected into the patient as required to
accomplish the control of the flow or presence of gastrin.
Another serious medical problem which is treatable by the
application of the technique of the instant invention is that of
hypertension. In general terms, the state of hypertension is the
abnormal level or fluctuation of one's blood pressure. The blood
pressure in an individual is controlled by many physiological
processes in the body. However, one major substance affecting the
regulation of such pressure is the hormonal polypeptide known as
angiotension II. In certain states of high blood pressure
(hypertension) it is difficult to medically control the secretion
and therefore the level of angiotension II in the circulatory
system. By the appropriate modification of this hormone and
subsequent immunization with this altered modified proteinaceous
hormone, it is possible to reduce the secretion of angiotension II
in patients with chronically elevated hormone levels. The
predictable and controlled reduction of this substance is
beneficial to certain patients with chronic problems of
hypertension. Modified angiotension II can be produced by the
general protein modification technique described herein. The
resultant modified angiotension II is simply injected into the
patient in an amount sufficient to induce antibody response
sufficient to control or regulate unmodified angiotension II to the
desired degree.
A further embodiment of the present invention is the treatment of
diabetes and associated micro and macro vascular diseases.
Currently, the treatment of diabetes is limited to dietary and/or
drug treatment to regulate blood glucose levels. Recent scientific
data support the concept that growth hormone and somatomedian (both
polypeptides) are intimately involved in the disease syndrome.
These substances can be modified by the technique described herein
and used in an effective amount to control the progress of this
disease. In practice, modified growth hormone or modified
somatomedian is injected into the body to develop antibodies for
control of the normally secreted hormones.
Another health problem that can be treated by the use of the
concepts of this invention is that of certain endocrine or hormone
dependent breast tumors or cancers. Certain of these cancers have
been shown to be dependent upon the abundant secretion of the
hormone prolactin for their continued survival. The inhibition of
the secretion of prolactin has been shown to diminish the growth
rate and the actual survival of certain of these tumors. The
immunization of such subjects with the hapten coupled or otherwise
altered prolactin produced as described herein, would result in the
systematic reduction of the level of this hormone circulating in
the system and consequently, may result in the regression or
remission of tumor growth. The consequence of this treatment would
be far more favorable in terms of effective treatment of this
disease since surgical removal of the breasts is a principal method
of treatment currently available. It should be understood that this
treatment should be effective for only those tumors that are
dependent upon the secretion of prolactin for survival.
Investigators also have determined, for example, that certain
polypeptide entities are supportive factors to and secretions of
neoplastic diseases in both man and other animals. These entities
have biochemically, biologically and immunologically close
resemblances to hormones, particularly to Chorionic Gonadotropin
(CG), as well as to Luteinizing Hormone (LH). By applying the
isoimmunization techniques of the invention, the function of such
polypeptides or endogenous counterparts can be neutralized to carry
out regulation of the malignancy. For example, tumors in both male
and female primates may be treated by isoimmunization procedures
developing antibodies to Chorionic Gonadotropin or Luteinizing
Hormone or the noted entity analogous thereto. Further, neoplasms
in primate females may be regulated by isoimmunization procedures
developing antibodies to endogenous Follicle Stimulating Hormone
(FSH). This hormone, when associated with a tumor state, tends to
aggravate the tumorous condition.
The immunochemical control asserted, as noted, neutralizes the
naturally occuring hormone or the above-described entity
biologically analogous thereto. As a consequence, the hormone or
entity will not be available as would normally be the case, for
example, the stimulation of some action of a target tissue.
Conversely, the neutralization of the biological activity of the
hormone or analogous entity may serve to take away an inhibitory
action which it otherwise might assert.
There are certain other disease states that may be treatable by the
use of altered or modified hormonal or non-hormonal proteins as
antigens. The disease states and the associated substances that may
be used as modified antigens for immunological treatment of these
diseases will be listed as follows:
(1) modified parathyroid hormone for the treatment of kidney
stones,
(2) modified insulin and/or glucagon for the treatment of
hyperinsulinoma,
(3) modified thyroid stimulating hormone (TSH) for the treatment of
hyperthyroidism, and
(4) modified secretin for the treatment of irritable bowel
syndrome.
Another group of polypeptides which can be altered by the
procedures described herein and used in the field of human
fertility control are specific non-hormonal protein antigens
isolated from placental tissue. There is direct evidence that
inhibition of substances that are specific to the placental tissue
and do not have similar antigenic properties with other antigens
from organs in other parts of the body, can result in the
disruption of pregnancies by passive immunization. Such specific
placental substances when modified to form modified polypeptides by
the procedures described herein can be injected into the body of an
animal of the same species as an effective fertility control means
with the mechanism being active immunization similar to that
described for the antigenic modification of hormones. The
particular advantage of these substances is that placental antigens
are foreign to the non-pregnant female human subject and therefore
are unlikely to cause any cross-reaction or disruption of normal
body function in the non-pregnant female.
While the invention is useful for the human species it will be
appreciated that it is also useful in connection with other
animals. Similarly, while the reference herein with respect to
fertility control is primarily directed to females, such described
techniques may be applicable to males, i.e. FSH, its beta subunit
and fragments thereof. Such immunization represents an effective
fertility control procedure, providing no physiological
consequences are encountered which may be found to react adversely
to the performance of other body constituents.
Whether the concerned hormone, non-hormonal protein or specific
fragment thereof which is modified is naturally occurring or is a
synthetic product is clearly immaterial. A synthetic protein
molecule will perform the same function as the naturally occurring
one, inasmuch as the body will react in an equivalent antigenic
manner.
It has accordingly been discovered by virtue of this invention that
it is possible to interfere with or treat various disease states or
medical problems which are caused or influenced by certain
polypeptides by active immunization of a male or female animal by
the reproduction and use of antigens formed by administration of
modified polypeptides. The modification of the polypeptides forms
antigens which are than administered into an animal in which
immunization is to be developed. Said modification is accomplished
by attaching to a polypeptide one or more foreign reactive
(modifying) groups and/or by attaching two or more polypeptides to
a foreign reactive group (i.e., a carrier) or both of the above, so
that the body of the animal, recognizing the modified polypeptide
as a foreign object, produces antibodies which neutralize not only
the modified protein but also the natural protein which is
responsible for the disease or medical problem being regulated. In
order to produce an effective quanta of antibodies to the antigen
or targeted functional polypeptide, it may be advantageous to
administer the modified polypeptide together with an immunological
adjuvant. The term "adjuvant" is commonly referred to by those
engaged in the field at hand as being a substance which will
elevate the total immune response of an animal or person to any
immunization thereof, i.e. the adjuvant is a nonspecific
immuno-stimulator.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a chart describing the results of mating four baboons
three times following the administration thereto of a fertility
controlling antigen according to the invention;
FIG. 2 shows two plots illustrating the antifertility antibody
levels maintained within two baboons following the administration
of antigens thereto formulated in accordance with the invention;
and
FIG. 3 shows three dose response lines illustrating the specificity
of antibody response to a CG antigen formulated in accordance with
the invention.
GENERAL DESCRIPTION
In an effort to better define the modified polypeptides with which
this invention is concerned, it is first considered appropriate to
set out more precisely than hereinabove, examples of the natural
hormones and natural non-hormonal proteins modified according to
this invention. They include Follicle Stimulating Hormone (FSH),
Luteinizing Hormone (LH), Chorionic Ganadotropin (CG), e.g. Human
Chorionic Ganadotropin (HCG), Placental Lactogen, e.g. Human
Placental Lactogen (HPL) Prolactin, e.g. Human Prolactin, (all of
which are proteinaceous reproductive hormones), gastrin,
angiotension II, growth hormone, somatomedian, parathyroid hormone,
insulin, glucagon, thyroid stimulating hormone (TSH), secretin, and
other polypeptides which could adversely affect body function.
The hormone, Chorionic Gonadotropin (CG) has been the subject of
extensive investigation, it being demonstrated in 1927 that the
blood and urine of pregnant women contained a gonad-stimulating
substance which, when injected into laboratory animals, produced
marked gonadal growth. Later, investigators demonstrated with
certainty that the Placental Chorionic villi, as opposed to the
pituitary, were the source of this hormone. Thus, the name
Chorionic Gonadotropin or, in the case of humans, Human Chorionic
Gonadotropin (HCG) was given to this hormone of pregnancy. During
the more recent past, a broadened variety of studies have been
conducted to describe levels of HCG in normal and abnormal
physiological states, indicating its role in maintaining pregnancy.
The studies have shown the hormones' ability to induce ovulation
and to stimulate corpus luteum function and evidence has been
evoked for showing its ability to suppress lymphocyte action. The
immunological properties of the HCG molecule also have been studied
widely. Cross-reaction of antibodies to HCG with human pituitary
Luteinizing Hormone (LH), and vice-versa, have been extensively
documented, see for example:
Paul , W. E. & Ross, F. T. (1964) Immunologic Cross Reaction
Between HCG and Human Pituitary Gonadotropin. Endrocrinology, Vol.
75, pp. 352-358.
Flux, D. X. & Li C. H. (1965) Immunological Cross Reaction
Among Gonadotropins. Acta Endrocrinologica, Vol. 48, pp. 61-72.
Bogshawe, K. D.; Orr, A. H. & Godden J. (1968) Cross-Reaction
in Radi-Immunoassay between HCG and Plasma from Various Species.
Journal of Endocrinology, Vol. 42, pp. 513-518.
Franchimont, P. (1970) Study on the Cross-Reaction between HCG and
Pituitary LH. European Journal of Clinical Investigation, Vol. 1,
pp. 65-68.
Dorner, M.; Brossmer, R.; Hilgenfeldt, U. & Trude, E. (1972).
Immunological reactions of Antibodies to HCG with HCG and its
chemical derivatives. In Structure-Activity Relationships of
Proteins and Polypeptide Hormones (ed. M. Margoulies & F. C.
Greenwood), pp. 539, 541 Amsterdam: Exerpta Medica Foundation.
Further, these cross-reactions have been used to perform
immunoassays for both CG and LM hormones. See:
Midgley, A. R. Jr. (1966) Radioimmunoassay: a method for HCG and
LH. Endocrinology, Vol. 79, pp. 10-16.
Crosignani, P. G., Polvani, F. & Saracci R. (1969)
Characteristics of a radioimmunoassay for HCE-LH. In Protein and
Polypeptide Hormones (ed. M. Margoulies) pp 409, 411 Amsterdam:
Excerpta Medica Foundation.
Isojima, S; Nake, O.; Kojama, K. & Adachi, H. (1970). Rapid
radioimmunoassay of human L.H. using polymerized anti-human HCG as
immunoadsorbent. Journal of Clinical Endocrinology and Metabolism,
Vol. 31, pp. 693-699.
In addition to providing for the modification of the entire hormone
or selected polypeptide, the invention further provides for the
utilization of modified subunits, for example the beta subunit of
Chorinonic Gonadotropin. Of particular interest, such subunits may
be fragmented into smaller components herein termed "fragments".
The latter can be produced synthetically to exhibit an amino acid
sequence sufficiently in analogous correspondence to a
predetermined portion of the parent subunit. Such fragments
generally are conjugated with a larger molecule or component
foreign to the body, which may be termed a "carrier", in order to
effectively evoke or raise a sufficient quanta of antibodies. The
use of the fragments, as thus conjugated, advantageously provides a
high degree of specificity of antigenic reaction to the targeted
hormone or its biochemical equivalent, i.e. the antibodies will not
react with other body constituents. Of particular interest, the
above-discussed cross reaction of HCG and LH can be avoided by
utilization of fragments of the respective hormone due to the
desirable specificity of response thereto. Thus, when interested in
obtaining an immunological reaction against the hormone, HCG, the
undesirable immune reaction to the naturally occuring body
constituent, LH, may be eliminated. Synthetic equivalents of the
fragments offer enhanced practicality both from the standpoint of
production costs and necessary maintenance of purity.
As is indicated in the above discussion, when considered in
isolation with respect to conception and pregnancy, CG only is
present in female primates when they are in a post conception
state. However, as discussed above and later herein, an entity at
least analogous thereto (having similar immunological properties to
HCG) is seen to be present in conjunction with malignancies.
Subunits and fragments of the proteinaceous reproductive hormones
include the beta subunit of natural Follicle Stimulating Hormone,
the beta subunit of natural Human Chorionic Gonadotropin, fragments
including, inter alia, a 20-30 or 30-39 amino acid peptide
consisting of the C-terminal residues of natural Human Chorionic
Gonadotropin beta subunit, as well as specific unique fragments of
natural Human Prolactin and natural Human Placental Lactogen, which
may bear little resemblance to analogous portions of other protein
hormones. Further with respect to the type of novel chemical
entities with which this invention is concerned, one may note for
instance the chemical configuration of the beta subunit of HCG.
That structure is as follows:
Ser-Lys-Glu-Pro-Leu-Arg-Pro-Arg-Cys-Arg.sup.10
-Pro-Ile-Asn*-Ala-Thr-Leu-Ala-Val-Glu-Lys.sup.20
-Glu-Gly-Cys-Pro-Val-Cys-Ile-Thr-Val-Asn*-Thr-Thr-Ile-Cys-Ala-Gly-Try-Cys-
Pro-Thr.sup.40 -Met-Thr-Arg-Val-Leu-Gln-Gly-Val-Leu-Pro.sup.50
-Ala-Leu-Pro-Gln-Val-Val-Cys-Asn-Try-Arg.sup.60
-Asp-Val-Arg-Phe-Glu-Ser-Ile-Arg-Leu-Pro.sup.70
-Gly-Cys-Pro-Arg-Gly-Val-Asn-Pro-Val-Val.sup.80
-Ser-Tyr-Ala-Val-Ala-Leu-Ser-Cys-Gln-Cys.sup.90
-Ala-Leu-Cys-Arg-Arg-Ser-Thr-Thr-Asp-Cys.sup.100
-Gly-Gly-Pro-Lys-Asp-His-Pro-Leu-Thr-Cys.sup.110
-Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Ser-Ser.sup.120
-Ser*-Lys-Ala-Pro-Pro-Pro-Ser*-Leu-Pro-Ser.sup.130
-Pro-Ser*-Arg-Leu-Pro-Gly-Pro-Ser*-Asp-Thr.sup.140
-Pro-Ile-Leu-Pro-Gln
Structure (I)
For specificity of antibody action it is necessary that distinctive
peptides be isolated or prepared that contain molecular structures
completely or substantially completely different from the other
hormones. The beta-subunit of HCG possesses a specific chain or
chains or amino acid moieties which differ either completely or
essentially from the polypeptide chain of Human Luteinizing
Hormone. These chains or fragments, when conjugated with a carrier,
represent an additional aspect of this invention. Accordingly, the
polypeptide Structures (II) and (III) [C-terminal portion of
structure I)]
Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Ser-Ser-Ser*-Lys-Ala-Pro-Pro-Pro-Ser*-Leu-P
ro-Ser-Pro-Ser*-Arg-Leu-Pro-Gly-Pro-Ser*-Asp-Thr-Pro-Ile-Leu-Pro-Gln
Structure (II)
Gln-Asp-Ser-Ser-Ser-Ser*-Lys-Ala-Pro-Pro-Pro-Ser*-Leu-Pro-Ser-Pro-Ser*-Arg-
Leu-Pro-Gly-Pro-Ser*-Asp-Thr-Pro-Ile-Leu-Pro-Gln
Structure (III)
whether obtained by purely synthetic methods or by enzymatic
degradation from the natural or parent polypeptide, [Carlson et
al., J. Biological Chemistry, 284 (19), p. 6810, (1973)] when
modified according to this invention, similarly provide materials
with antigenic properties sufficient to provide the desired
immunological response. It will be understood, for example, that
addition of a polytyrosine chain or a protein macromolecule
(carrier) may assist in rendering Structure (II) antigenic so that
the resulting administration of modified Structure (II) will
provide the desired immunological action against natural HCG.
The beta subunit set forth at Structure (I) is seen to represent a
chemical sequence of 145 amino acid components. This structure has
a high degree of structural homology with the corresponding subunit
of Luteinizing Hormone (LH) to the extent of the initial 110 amino
acid components. As indicated above, it may be found desirable,
therefore, to evoke a high specificity to the Chorionic
Gonadotropin hormone or an analogous entity through the use of
fragments analogous to the C-terminal, 111-145 amino acid sequence
of the subunit. Structure (II) above may be observed to represent
just that sequence. Structure (III) is slightly shorter,
representing the 116-145 amino acid positions within the subunit
sequence.
Further polypeptide chains useful in promoting antibody buildup
against natural HCG include the following structures labeled
Structures (IV) through (XIV). When modified according to this
disclosure, such as by coupling to Ficoll 70* or other
modifier-carriers such as protein macromolecules described herein,
these polypeptides provide immunogenic activity with which this
invention is concerned. All of these polypeptides are considered
fragments of HCG by virtue of their substantial resemblance to the
chemical configuration of the natural hormone and the immunological
response provided by them when modified as indicated herein.
Cys-Pro-Pro-Pro-Pro-Pro-Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln
Structure (IV)
Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Pro-Pro-Pro-Pro-Pro-Pro-Cys
Structure (V)
Phe-Gln-Asp-Ser-Ser-Ser-Ser-Lys-Ala-Pro-Pro-Pro-Ser-Leu-Pro-Ser-Pro-Ser-Arg
-Leu-Pro-Gly-Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln
Structure (VI)
Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Ser-Ser-Ser-Lys-Als-Pro-Pro-Pro-Ser-Leu-Pro
-Ser
Structure (VII)
Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Pro-Pro-Pro-Cys-Pro-Pro-Pro-Ser-Asp-Thr-Pro
-Ile-Leu-Pro-Gln
Structure (VIII)
Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Pro-Pro-Pro-Pro-Pro-Pro-Cys-Pro-Pro-Pro-Pro
-Pro-Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln
Structure (VIIIa)
Asp-His-Pro-Leu-Thr-Aba-Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Ser-Ser-Ser-Lys-Als
-Pro-Pro-Pro-Ser-Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pro-Gly-Pro-Ser-Asp-Thr-Pro-Il
e-Leu-Pro-Gln-Pro-Pro-Pro-Pro-Pro-Pro-Cys
Structure (IX)
Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Ser-Ser-Ser-Lys-Ala-Pro-Pro-Pro-Ser-Leu-Pro
-Ser-Pro-Ser-Arg-Leu-Pro-Gly-Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln-Pro-Pro-Pr
o-Pro-Pro-Pro-Cys
Structure (X)
Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Ser-Ser-Ser-Lys-Ala-Pro-Pro-Pro-Ser-Leu-Pro
-Ser-Pro-Ser-Arg-Leu-Pro-Gly-Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln-Cys
Structure (XI)
Structure (IV) will be recognized as incorporating a Cys component
at the amino or N terminal which is associated with a Proline
spacer sequence. These spacers serve to position the sequence which
follows physically distant from the carrier-modifier. The latter
sequence may be observed to represent the 138th to 145th amino acid
component sequence of the subunit Structure (I). Structure (V) on
the other hand, represents an initial sequence corresponding with
the 111th to 118th components of the subunit structure (I) followed
by a sequence of six Proline spacer components and a carboxyl
terminal, present as Cysteine. The rationale in providing such a
structure is to eliminate the provision of sites which may remain
antigenically neutral in performance. Structures (IV) and (V)
represent relatively shorter amino acid sequences to the extent
that each serves to develop one determinant site. Consequently, as
alluded to in more detail hereinafter, they are utilized in
conjunction with a mixed immunization technique wherein a necessary
two distinct determinants are provided by the simultaneous
administration of two such fragments, each conjugated to a
corresponding, separate carrier macromolecule. Structure (VI)
represents the 115th through 145th component sequence of structure
(I). Structure (VII) represents a portion of Structure (I),
however, essentially, a sequence of the 111th to 130th components
thereof is formed.
Structure (VIII) incorporates two sequences, one which may be
recognized in Structure (V) and the other in Structure IV. These
two sequences are separated by two spacer sequences of Proline
components and one is joined with an intermediately disposed
Cysteine component which serves a conjugation function as described
later herein. With the arrangement, two distinct determinant sites
are developed in physically spaced relationship to avoid the
development of an unwanted artificial determinant possibly
otherwise evolved in the vicinity of their mutual coupling.
Structure (VIIIa) represents Structure (VIII) with additional Pro
spacer residues to provide a widened spacing of determinant
sites.
Structure (IX) mimics sequences from Structure (I) with the
addition of a Proline Spacer Sequence, A Cysteine Component at the
C-terminal, and an Aba substituted for Cys at the 110 position. The
Aba designation is intended herein to mean alpha-aminobutyric acid
of Cysteine. Structure (X) will be recognized as a combination of
Structure (II) with a six residue Proline spacer sequence and a
Cysteine component at the C-terminal. Similarly, Structure (XI)
combines Structure (II) with a Cysteine component at the C-terminal
without a Proline spacer sequence.
Thr-Cys-Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Ser-Ser-Ser-Lys-Ala-Pro-Pro-Pro-Ser
-Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pro-Gly-Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln
Structure (XII)
Asp-His-Pro-Leu-Thr-Aba-Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Ser-Ser-Ser-Lys-Ala
-Pro-Pro-Pro-Ser-Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pro-Gly-Pro-Ser-Asp-Thr-Pro-Il
e-Leu-Pro-Gln-Cys
Structure (XIII)
Cys-Pro-Pro-Pro-Pro-Pro-Pro-Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Ser-Ser-Ser-Lys
-Ala-Pro-Pro-Pro-Ser-Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pro-Gly-Pro-Ser-Asp-Thr-Pr
o-Ile-Leu-Pro-Gln
Structure (XIV)
Structure (XII) will be recognized as having the sequence of
Structure (II) with the addition of Thr-Cys components at its N
terminal. Structure (XIII) is similar to structure (IX) but does
not contain the spacer conjugate. Structure (XIV) will be
recognized as being similar to Structure II with the addition of
spacer components at the N terminal and a Cys component for
conjugation purposes.
Particularly where the larger whole hormone or subunit type
molecular structures are concerned, the number of foreign reactive
groups which are to be attached to the polypeptide and the number
of polypeptides to be attached to a foreign reaction group depends
on the specific problem being treated. Basically, what is required
is that the concerned polypeptide be modified to a degree
sufficient to cause it to be antigenic when injected in the body of
the host. If too little modification is effected, the body may not
recognize the modified polypeptide as a foreign body and would not
create antibodies against it. If the number of foreign molecules
added to the polypeptides is too great, the body will create
antibodies against the intruder antigen, but the antibodies will be
specific to the injected antigen and will not neutralize the action
of the concerned natural endogenous hormone or non-hormonal
protein, i.e. they will be specific to the modifier.
In general, again considering the larger molecule subunit or whole
hormone, it has been found that about 1-40 modifying groups per
molecule of polypeptide will be useful in modifying the polypeptide
adequately so as to obtain the desired immunological effect of this
invention. As will be appreciated by one skilled in the art, this
ratio of modifying groups per polypeptide will vary depending upon
whether an entire hormone is utilized for modification or whether
for instance a relatively small synthetic fragment of said hormone
is to be modified. Generally for the larger molecules, it is
preferred that 2-40 modifying groups per molecule of polypeptide be
used according to this invention. In the instance where the
polypeptide is the .beta.-subunit of HCG it is particularly
preferred that about 5-30 and more preferably 10-26 modifying
groups per molecule of polypeptide be used. The important
consideration with respect to each modified polypeptide is that the
degree of modification be adequate to induce generation by the body
of antibodies adequate to neutralize some of the natural hormone or
non-hormonal protein against which neutralization is desired, and
this will vary with each polypeptide involved, and the degree of
correction or change desired for the body function involved.
Modification of the polypeptide is accomplished by attaching
various kinds of modifying groups to proteinaceous hormone,
non-hormonal proteins, subunits or specific fragments thereof
according to methods known in the art.
As is apparent, structures (II)-(XIV) are relatively smaller
fragments, usually produced synthetically. To render them capable
of eliciting antibody production, it becomes necessary to conjugate
them with larger carrier-modifier molecules. Generally about 5-30
peptide fragments will be coupled with one carrier molecule. The
body will, in effect, recognize these foreign carriers as well as
the sequences represented by the fragments and form antibodies both
to the carrier and to the sequences of the coupled fragments. Note
that the carrier-modifiers are foreign to the body and thus
antibodies to them will not be harmful to any normal body
constituents. In the latter regard, it may be found preferable to
utilize a carrier which, through the development of antibodies
specific to it, will be found beneficial to the recipient.
As indicated earlier herein, it also is preferred that the
modification constitute two or more immunological determinants
represented on the native hormone as polypeptide structures to
which it is desired to evoke an antibody response. The effect is
one of heterogenity of antibody development. Thus, several fragment
structures have been described above having at least two distinct
amino acid sequences represented in the HCG beta subunit [Structure
I]. These sequences may be so spaced as to derive the determinants
in mutual isolation, while the spaced sequence fragment is
conjugated with a larger, macromolecular carrier. Alternately, the
noted mixed immunization arrangement may be utilized wherein a
first fragment developing one determinant is conjugated with a
first carrier molecule and is administered in combination with a
second, distinct fragment which is conjugated with a second carrier
molecule, the latter of which may be the same as or different in
structure from the first carrier. Thus, each macromolecular carrier
must be conjugated with hormone fragments such that each fragment
represents two or more immunological determinants. These two
necessary determinants can be evolved by mixing, for example,
separate conjugate structures, for example Structures (IV) and (V)
each of which, through forming antibodies separately to the
distinct determinants, will provide a population of antibodies
reacting with two separate determinants on the natural endogenous
hormone.
Inasmuch as the noted fragments are relatively small as compared,
for instance, to a whole hormone or subunit thereof, a criterion of
size is imposed upon the selection of a carrier. The carrier size
must be adequate for the body immune system to recognize its
foreign nature and raise antibodies to it. Additionally, carrier
selection preferably is predicated upon the noted antibody
heterogeneity requirement, i.e. the carrier must itself evoke a
heterogeneous immune response in addition to the fragments. For
example, improved response may be recognized where the carrier is
varied in structure, e.g. incorporating branching chains to enhance
the recognition of both the carrier and the attached polypeptide as
being of a foreign nature.
As one example of whole hormone modification, modified diazo groups
derived from sulfanilic acid may be attached to the subject
polypeptides, see the Cinader et al and Phillips et al references
cited subsequently for instruction on how this "attachment" is
accomplished, and to the extent necessary for an understanding of
this invention, such is incorporated herein by reference.
Additional modifying groups for modifying whole hormones or their
subunits are those groups obtained by reaction of the polypeptides
with dinitrophenol, trinitrophenol, and S-acetomercaptosuccinic
anhydride, while, suited for utilization as a carrier-modifier in
conjunction with fragments, are polytyrosine in either straight or
branched chains, polyalanines in straight or branched chains,
biodegradable polydextran, e.g. polymerized sugars such as sucrose
copolymerized with epichlorohydrin, e.g. Ficoll 70 and Ficoll 400*
or a polyglucose such as Dextran T 70**, serum proteins such as
homologous serum albumin, hemocyanin from Keyhole limpet, a marine
gastropod mollusk, viruses such as influenza virus (type A, B, or
C) or poliomyelitis virus, live or killed, Types 1, 2 and 3 of
tetanus toxoid, diphtheria toxiod, cholera organisms or somewhat
less preferably, natural proteins such as thyroglobulin, and the
like. Generally, synthetic modifiers are preferred over the natural
modifiers. However, carrier-modifiers found particularly suitable
for conjugation with the above-discussed fragment structures are
Flagellin, tetanus toxoid and an influenza subunit, for example,
the preparation of which is described by Bachmeyer, Schmidt and
Liehi, "Preparation and Properties of a Novel Influenza Subunit
Vaccine", Post-Graduate Medical Journal (June, 1976) 52:360-367.
This influenza subunit was developed as a vaccine which
incorporates essentially only the two viral proteins,
Haemagglutinin and Neuraminidase. Containing substantially only
these two essential immunogens, the subunit represents a
preparation which does not contain other protein and lipid antigens
which may be found to cause undesired side reactions. A secondary
benefit may be realized through the utilization for example, of the
influenza subunit, poliomyelitis virus, tetanus toxoid, diphtheria
toxoid, cholera antigens or the like as a modifier-carrier,
inasmuch as beneficial antibodies will be raised to that
modifier-carrier as well as to the hormonal fragment conjugated
thereto.
Flagellin is a protein described as forming the wall of the main
spiral filament of the flagellum. Bacterial flagella, in turn, have
been known as the active organelles of cell locomotion, individual
flagella (flagellum) occurring in suspension as individual spirals
which, upon drying, collapse into filaments which describe a sine
wave with a wave length of 2-3 microns and an amplitude of
0.25-0.60 microns. Generally, the flagellum consists of three
morphologically distinct parts: a basal structure that is closely
associated with the cytoplasmic membrane and cell wall, a hook and
the noted main spiral filament.
Purified flagellum is readily obtained by solubilization of
flagellar filaments below a pH value of about four, and subsequent
removal of the insoluble material by centrifugation or filtration.
As a group of related proteins, flagellins from different bacterial
species have been predicted to have similar amino-acid
compositions. However, the amino acid composition of each flagellin
species is unique. Essentially all flagellins are described as
containing no or only a few residues of cysteine, tryptophan,
tyrosine, proline and histidine. Thus, when conjugated with
fragments in accordance with the invention, a thiolactonization
procedure or the like is carried out as described later herein.
The molecular weights of various flagellin have been calculated, in
all cases the values thereof of the monomeric subunits falling in
the range of 30,000 to 50,000. From an immunological standpoint, a
flagellin molecule is highly immunogenic. For a further and more
detailed discourse describing bacterial flagella and flagellin,
reference is made to "Advances in Microbial Physiology" 6:219 1971,
"Bacterial Flagella" by R. W. Smith and Henry Coffler, which
publication is incorporated herein by reference.
Tetanus toxoids have been the subject of study and production for
many years. The toxoid generally is evolved from a formalinization
of tetanus toxin, the latter being a protein synthesized by
Clostridium tetani. Immunization currently is carried out utilizing
soluble and absorbed tetanus toxoids and suggestions have been made
concerning the utilization of fluid tetanus toxoid in complex with
antitoxin. Publications describing the toxin and toxoid are
numerous, reference being made to the following:
1. Immunochemistry of Tetanus Toxin, Bizzini, et al, Journal of
Biochemistry, Vol. 39, pp. 171-181 (1973).
2. Early and Enhanced Antitoxin Responses Elicited with Complexes
of Tetanus Toxoid and Specific Mouse and Human Antibodies, Stoner
et al, Journal of Infectious Diseases, Vol. 131, No. 3, pp. 230-238
(1975).
3. Differences in Primary and Secondary Immunizability of Inbred
Mice Strains, Ipsen, Journal of Immunology, Vol. 83, pp. 448-457
(1959);
4. Antigenic Thresholds of Antitoxin Responses Elicited in
Irradiated Mice with Complexes of Tetanus Toxin and Specific
Antibody, Hess et al, Radiation Research, Vol. 25, pp. 655-667
(1965).
5. Early and Enhanced Germinal Center Formation and Antibody
Responses in Mice After Primary Stimulation with Antigenisologous
Antibody Complexes as Compared with Antigen Alone, Laissue et al,
Journal of Immunology, Vol. 107, pp. 822-825, (1971).
6. Distinctive Medullary and Germinal Center Proliferative Patterns
in Mouse Lymph Nodes after Regional Primary and Secondary
Stimulation with Tetanus Toxoid, Buerki et al, Journal of
Immunology, Vol. 112, No. 6, pp. 1961-1970 (1974)
Modification by removal of moieties is also contemplated by this
invention. Thus, for example, where certain of the natural proteins
have carbohydrate moieties, these carbohydrate moieties may be
removed according to methods known in the art by, for instance,
N-acetyl neuriminidase or N-acetyl glucosidase, materials useful
for removal of specific carbohydrate moieties.
These various means for modification are, as indicated above, known
to persons skilled in the art. Certain of these means may be found
in the following list of literature references, whereas various
others of them may be found elsewhere in the literature by
art-skilled persons:
(1) Klotz et al., Arch. of Biochem. and Biophys., 96, pp. 605-612,
(1966).
(2) Khorana, Chem. Rev. S3: 145, (1953).
(3) Sela et al., Biochem. J., 85, p. 223, (1962).
(4) Eisen et al., J. Am. Chem. Soc. 75, 4583, (1953).
(5) Centeno et al., Fed. Proc. (ABSTR) 25: 729, (1966).
(6) Sokolowsky et al., J. Am. Chem., Soc. 86: 1212, (1964).
(7) Tabachnick et al., J. Biol. Chem. 234, No. 7, p. 1726,
(1959).
(8) Crampton et al., Proc. Soc. Exper. Biol. & Med. 80: 448,
(1952).
(9) Goodfriend et al., Science 144, p. 1344 (1964).
(10) Sela et al., J. Am. Chem. Soc., 78, p. 746, (1955).
(11) Cinader et al., Brit. of Exp. Pathol. 36, p. 515, (1955).
(12) Phillips et al, J. of Biol. Chem. 240 (2), pp. 699-704,
(1965).
(13) Bahl, J. of Biol. Chem., 244, p. 575, (1969).
Methods for preparing the modified polypeptides of this invention
also include the following.
In one preferred modification approach, the polypeptide fraction,
for example, structure (XII), is activated first following which it
is conjugated with a carrier, for example the influenza subunit
described above, tetanus toxoid or Flagellin. An activating reagent
may be utilized which exhibits differing functionality at its ends
and by choice of reaction conditions, these end components can be
made to react selectively. For example, the following activators A
and B, having a maleiimido group and a substituted acid group, may
be provided: ##STR1## where X is a non-reacting group made up of a
substituted, or unsubstituted phenyl or C.sub.1 -C.sub.10 alkalene
moiety, or a combination thereof. In this regard, the moiety
substituted on the phenyl should be non-interfering as is the
remainder of the "X" grouping. X may, inter alia, be selected from
the following: ##STR2##
The maleiimido grouping of the above reagents will react with
sulfhydryl (SH) groups in the polypeptide fragments under
conditions whereby the opposite end (active ester end) of the
reagent does not react with the amino groups present in the
fragment sequences. Thus, for example, polypeptide fragments such
as structure (XII), containing a Cys amino acid and hence, as SH
group react as follows: ##STR3## Following the above, upon
adjusting the pH to a slightly alkaline condition, e.g. 8, and
adding the carrier protein accomplishes the following conjugation:
##STR4##
Alternately, a carrier protein such as the above-noted Flagellin
which does not contain SH groups, but does contain NH.sub.2 groups,
may first be treated with activator A or B at pH 7 or lower at the
active ester end, giving: ##STR5## Following the above, the
activated carrier is reacted with a polypeptide fragment containing
an SH group to derive a product similar to that discussed
immediately above.
Should the polypeptide fragment not contain an SH group, e.g.
Structures (II), (III), (VI) and (VII), such structures can be
modified first to introduce such a grouping by standard methods
such as "thiolactonization", following which they are conjugated
utilizing the above-discussed selective bi-functional reagents. For
a more detailed description of these reagents, reference is made to
the following publications:
O. Keller and J. Rudinger, Helv. Chim. Acta 58, 531-541 (1975).
W. Trommer, H. Kolkenbrock & G. Pfleiderer, Hoppe-Seyler's Z.
Physiol. Chem., 356, 1455-1458 (1975).
Further description of the preferred embodiments of the
above-described utilization of bi-functional reagents is provided
hereinbelow at Examples XXVII and XXVIII.
As an alternate approach to the utilization of the maleiimido group
reagents discussed above, an alkylation step may be used to cause
conjugation. Conditions can be chosen such that in the presence of
amino groups, essentially only SH groups will be alkylated. With
this approach, a generalization of the reactions carried out may be
expressed as follows: ##STR6##
With this approach, the larger molecule carrier, e.g. Flagellin,
tetanus toxiod or the influenza subunit described herein is first
modified by reaction of a fraction of its amino groups with an
active ester of chloro, dichloro, bromo or iodo acetic acid, such
as: ##STR7## and this modified carrier is then reacted with the
sulfhydryl group in a polypeptide fraction, or a polypeptide
fraction which has been modified to contain the SH group (e.g.
thiolactonization) if it does not already have such a group. Such
modification is described in Example XXV below. The present
approach produces a thio ether linkage by alkylation of a free
thiol (sulfhydryl group).
With the instant procedure, the roles of the fragment and carrier
may be reversed, the fragment being modified to contain the
halomethyl alkylating group which would then react with sulfhydryl
groups in the carrier, or a carrier suitably modified to exhibit a
sulfhydryl group. More description of this selective alkylation of
sulfhydryl groups is provided in conjunction with Example XXX
below.
It may be seen from an observation of the formulae of Structures
(IV), (V), (IX), (X), (XI), (XII), (XIII) and (XIV) that a Cys
amino acid, which in a reduced state provides an SH reactive group,
is located at either the C terminal or N terminal of the peptide
structure. This location permits the peptide to be chemically
linked to carrier molecules at either terminus. And some Structures
(XIV), (X), (IX), (X), (IV) have a six-Prolene spacer chain
(Pro).sub.6 between the Cys residue and the remainder of the
peptide sequence. This latter arrangement provides a chemical
spacer between the coupled carrier and the sequences representing a
fragment of the natural hormone. A six-Prolene spacer can be added
as a side chain spacer, for example at position 122 (Lys) in
Structure (II), by initially adding an SH group (thiolactonization)
to the free or unblocked epsilon amino group on this (Lys) residue,
as set out in Example XXIX below. Then, utilizing the activator A,B
above in which the component "X" is a chain of six Prolene amino
acids, conjugation can be carried out. In the latter case, a spacer
is provided between the carrier and peptide linked at an
intermediate site, for example at position 122 in structure II. In
the former case, only the space represented by conjugating reagent
links the carrier and peptide.
Modifying groups, such as hemocyanin from Keyhole limpet,
containing free amino groups, are prepared in buffer solution such
as phosphate buffer, in sodium chloride solution at a pH of 6-8. To
this solution, tolylene diisocyanate (T.D.I.C.) reagent diluted
from about 1-10 to about 1-40 times with dioxane, is added to the
modifying group. The general procedure was disclosed by Singer and
Schick, J. Biophysical and Biochem. Cytology 9:519 (1961). The
amount of T.D.I.C. added may range from 0.075 to 1,000 molar
equivalents of the modifier used. The reaction may be carried out
at about -5.degree. to about +10.degree. C., preferably 0.degree.
to 4.degree. C., for about 1/2 to 2 hours. Any excess T.D.I.C. may
be removed by centrifugation. The precipitate may be washed with
the above-mentioned phosphate buffer and the supernatants
combined.
This activated modifying group solution may then be combined with
the hormonal or non-hormonal polypeptide to be conjugated.
Polypeptide is dissolved in the same phospate buffer (5-30 mg/ml)
and the volume of modifier and polypeptide combined according to
the molar ratio of the two desired in the conjugate. Combined
solutions are reacted at 30.degree.-50.degree. C., preferably
35.degree.-40.degree. C., for 3-6 hours.
Separation of modified polypeptide and free unconjugated
polypeptide may be accomplished by conventional techniques, such as
gel filtration.
Picogram amounts of I.sup.125 labeled polypeptide may be added as a
tracer to the reaction mixture at the time of conjugation, and a
quantity of polypeptide conjugated to modifying groups (molar
ratio) may be determined by the amount of radioactivity
recovered.
Included in the methods for modifying the hormones, non-hormonal
proteins and their fragments (unmodified polypeptides) are
conjugation by use of water-soluble carbodiimide. The amino groups
of the unmodified polypeptide are first preferably protected by
acetylation. This (acetylated) unmodified polypeptide is then
conjugated to modifier, such as natural protein modifier, e.g.
hemocyanin from Keyhole limpet, homologous serum albumin, and the
like, or Dextrans, Ficolls, or polytyrosine, preferably in the
presence of guanidine such as guanidine HCl, using 10-ethyl-3
(3-dimethylamino propyl) carbodiimide as activating agent. This
method is generally disclosed by Hoare and Koshland, Jr., J. of
Biological Chemistry 242:2447 (1967). In the instance where Ficoll
70 is used, it is preferred that it be first treated with ethylene
diamine so as to render the final coupling more efficient. This
treatment with ethylene diamine may be performed in solvent such as
saline and dioxane at about room tempreature and a pH of about
9-12, preferably 10-11 for about 1/4 to about 2 hours. The
conjugation itself between the unmodified polypeptide and the
modifier may be performed in solvent such as glycine methyl ester
while maintaining the pH at about 4-5, preferably about 4.5-4.8.
The temperature of reaction is conveniently about room temperature
and the reaction may be allowed to proceed for about 2-8 hours,
preferably 5 hours. The resulting modified polypeptide with which
this invention is concerned may be purified by conventional
techniques, such as column chromatography.
The immunogenic substances for this invention may also be provided
by polymerization of unmodified polypeptide using bifunctional
imidoester. The imidoester, such as dimethyl adipimidate, dimethyl
suberimidate and diethyl malonimidate, may be used to form the
polymer in a manner similar to the generally described methods of
Hartman and Wold; Biochem. 6:2439 (1967). The polymerization may
take place conveniently at room temperature in aqueous solvent at a
pH of about 9-12, preferably about 10-11, over a period of 1/4-2
hours.
Said immunogenic substances may also be prepared by dimerization
through a disulfide bond formed by oxidation of the thiol group on
a Cys-residue using iodosobenzoic acid and methods corresponding to
known methods, such as room temperature reaction for about 10-40
minutes.
Modified polypeptides may also be prepared using glutaric
dialdehyde as conjugating agent. According to a theory proposed by
Richards and Knowles [J. Mol. Biol. 37:231 (1968)], commercial
glutaric dialdehyde contains virtually no free glutaric dialdehyde,
but rather consists of a very complex mixture of polymers rich in
.alpha., .beta.-unsaturated aldehydes. Upon reaction with natural
protein modifiers such as homologous serum albumins, these polymers
form a stable bond through the free amino group, leaving aldehyde
groups free. This intermediate product then reacts with unmodified
polypeptide in the presence of alkali metal borohydride, such as
sodium borohydride. This intermediate is formed at pH 7-10,
preferably 8-9, at about room tempreature. The modified polypeptide
is also conveniently obtained at about room temperature after about
1/4-2 hours' reaction time. The resulting product is recovered in
pure form by conventional techniques, such as gel filtration,
dialysis and lyophilization.
Polymerized sugar modifiers such as Ficoll 70 or Dextran T 70 may
also be prepared for conjugation by treatment with a cyanuric
halide such as cyanuric chloride to form a dihalotriazinyl adduct.
The process may be performed in solvent such as dimethylformamide
at about 0.degree.-20.degree. C., preferably 10.degree.-15.degree.
C., for about 1/2-4 hours. The resulting intermediate product may
then be dialyzed until essentially halogen ion free, and
lyophilized and treated with unmodified polypeptides at pH 8-11,
preferably about 9-10, for about 1/2-12 hours at about
15.degree.-35.degree. C., conveniently at room temperature. The
resulting modified polypeptide may be recovered as indicated
above.
Said polymerized sugar modifiers may also be treated with alkali
metal periodate, such as sodium periodate, at a pH of 3-6 at about
30.degree.-60.degree. C. for about 1/2-4 hours, and the resulting
intermediate conjugated with unmodified polypeptide at a pH of
about 7-11, preferably about 8-10, for about 1/4 to about 2 hours
at a temperature of about 15.degree.-80.degree. C., preferably
20.degree.-60.degree. C. The resulting immunogenic substance
according to this invention may be separated as indicated
previously.
The modifying groups may vary in chemistry and number for any given
polypeptide structure. However, they will attach to only certain
amino acid moieties. In particular, when modifying with diazo
groups they will chemically bond to only the histidine, arginine,
tyrosine and lysine moieties or sites. Other modifying groups will
bond to peptide molecules at different sites and in different
numbers. Consequently, depending upon the size and chemical make-up
of a particular modified polypeptide desired, one skilled in the
art will readily be able to calculate the maximum possible number
of modifying groups associable with a polypeptide. It is also
recognized that several modifying groups may attach themselves to
each other which in turn attach to a single amino acid moiety, but
as used herein, reference to a number of modifying groups means the
number of reaction sites to which a modifier has been attached.
As indicated above, a theory leading to this invention was that the
chemical modification of an essential reproductive hormone would
alter it such that it would exhibit antigenic properties so that
when injected into an animal (including humans) it would cause the
formation of antibodies which in turn would not only react to the
injected modified hormone but also to the natural unmodified
endogenous hormone as well. With this theory in mind, reproductive
hormones of various species were modified and tested in baboons.
The results illustrated that modified hormones of unrelated species
do not produce the desired results, whereas modified hormones of
the same or closely related species do produce the desired results.
It will accordingly be clear that the polypeptide to be modified
should be so related to the endogenous hormone or non-hormonal
protein as to be either from the same animal species or be the
immunological equivalent thereof as modified.
Additional experiments were conducted to test the validity of this
concept in humans, i.e. modified human reproductive hormones
injected into humans. Collectively, the results prove the
conclusion drawn from the experiments with the baboons, namely,
isoantigenic immunization using modified human reproductive
hormones does produce contraception or interruption of gestation.
Detailed examples which follow illustrate this result.
It is known that fragments of endogenous hormones exhibit
essentially no antigenic properties. However, should a large enough
fragment of an endogenous hormone be slightly modified as indicated
above, then antibodies will be formed which will react in the same
way as if the modification is on a whole hormone, provided the
large fragment is sufficiently distinctive in chemical and physical
make-up as to be recognized as a specific part of the whole.
Whether the hormone or specific fragment thereof is naturally
occurring or is a synthetic product is clearly immaterial. A
synthetic hormone molecule will perform the same function as the
naturally occurring one, being equivalent for the purpose of this
invention. In this connection, it will be noted that natural
substances with which this invention is concerned possess
carbohydrate moieties attached at certain sites thereof whereas the
contemplated corresponding synthetic polypeptides do not.
Nevertheless, for the purpose of the instant specification and
claims, the synthetic and natural polypeptides are treated as
equivalents and both are intended to be embraced by this invention.
Reference in the above regard is made to Table No. 3 herein as read
in conjunction with Example XXIX.
Thus, where the word "hormone" or "hormone molecule" is used
herein, the word "synthetic" may be added before "hormone" without
changing the meaning of the discussion. Similarly, the word,
"fragment" may be inserted after "hormone" or "molecule" without
changing the meaning, whether or not "synthetic" has been inserted
before "hormone".
Throughout the above specification, the term "modified" has been
utilized in referring to the chemical reaction by which the foreign
molecules become chemically attached to specific sites on the
usually much larger polypeptide molecule. Although specific
mechanisms by which this is accomplished are described herein in
detail, other appropriate mechanisms may be used if desired. It is
clear that the modifier, i.e., the substance which modifies the
concerned protein, can be a physically larger molecule or fragment
thereof than the molecule or fragment which it modifies. As noted
above, such large molecules are deemed herein to be "carriers".
Clearly, physical size of the fragment is not always critical; the
criterion for effectiveness being that the body reaction generate
antibodies in sufficient quanta and specific to the targeted
hormone or endogenous substance.
The modified polypeptides of this invention may be administered
parenterally to the animals to be protected, preferably with a
pharmaceutically acceptable injectable vehicle. They may be
administered in conventional vehicles with other standard
adjuvants, as may be desirable, in the form of injectable solutions
or suspensions. As indicated earlier, the adjuvant serves as a
substance which will elevate total immune response in the course of
the immunization procedure. Lipasomes have been suggested as
suitable adjuvants. The soluble salts of aluminum, that is,
aluminum phosphate or aluminum hydroxide, have been utilized as
adjuvants in routine clinical applications in man. Bacterial
endotoxins or endotoxoids have been used as adjuvants as well as
polynucleotides and polyelectrolytes and water soluble adjuvants
such as muramyl dipeptides. The adjuvants developed by Freund have
long been known by investigators, however, the use thereof is
limited to non-human experimental procedures by virtue of a variety
of side effects evoked. The preferred mode of administration of the
entire vaccine is intramuscular.
The amount of modified polypeptide to be administered will vary
depending upon various factors, including the condition being
treated and its severity. However, in general, unit doses of 0.1-50
mg in large mammals administered one to five times at intervals of
one to five weeks provide satisfactory results. Primary
immunization may also be followed by "booster" immunization at one
to twelve month intervals.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
EXAMPLE I
Adult female baboons were studied for at least one menstrual cycle
for patterns of urinary estrogens, plasma, progestin, and in some
cases urinary LH. Only those animals displaying normal patterns of
these hormones were immunized. The criteria for normality and the
procedures for housing animals are well known and will not be
described.
Gonadotropin Preparations
Human Luteinizing Hormone (HLH)--partially purified preparation
from human pituitaries with a biological potency of 2.5 units per
mg. (NIH-LH-SI).
Human Follicle Stimulating Hormone (HFSH)--a partially purified
preparation from human pituitaries with a biological potency of 86
units per mg. (NIH-FSH-SI).
Human Chorionic Gonadotropin (HCG)--a highly purified preparation
from human pregnancy urine with biological potency of 13,200 IU/mg.
(2nd IRP-HCG).
Monkey Luteinizing Hormone (MLH)--a crude preparation from rhesus
monkey pituitaries with a biological potency of 0.75 units per mg.
(NIH-LH-SI).
Ovine Luteinizing Hormone (OLH) (NIH-LH-S5).
Baboon Luteinizing Hormone (BLH)--partially purified baboon
pituitary preparation with a biological potency of 1.1 units per
mg. (NIH-LH-S1).
All preparations, excepting the OLH, were prepared in the
inventor's laboratory. LH and HCG biological activity was
determined by the ovarian ascorbic acid depletion test and the FSH
preparation assayed by the ovarian augmentation assay.
Hormones were altered as antigens by coupling with a hapten in
varying ratios of hapten to hormone as described by Cinader et al.,
supra. For convenience, the Cinader process is discussed herein
although Phillips, supra, may provide a more stable bond under
certain circumstances. In this procedure, the protein hormone
serves as a carrier and the hapten is coupled to it by diazo bonds.
Although a variety of hapten groups were coupled to different
hormones, the same basic procedure was used for any combination.
Fifteen to thirty-five haptenic groups per hormone molecule were
found most useful for preparing immunizing antigens. The basic
reaction consisted of diazotizing the hapten (sulfanilic acid) by
adding it to a solution of 0.11 N HCl and then slowly adding this
solution dropwise to a 1 percent solution of NaNO.sub.2 with
constant stirring at 4.degree. C. Diazotization was considered
complete when free HNO.sub.2 was detected in the reaction mixture.
Although the above reaction was accomplished at 4.degree. C.,
optimum temperatures for the reaction normally are about
0.degree.-6.degree. C., although 4.degree. C. is preferred.
The hapten-protein coupling was performed by dissolving the protein
hormone in an alkaline buffer, pH 8.0. The diazotized hapten was
added slowly to the hormone solution with continuous stirring at
4.degree. C. The pH of the reaction was constantly monitored and
kept near 8.0. After all the hapten was added, the pH was finally
adjusted to 8.0, stirred for 1-2 hours and allowed to stand at
4.degree. overnight. The mixture was thoroughly dialyzed for 6-8
days against distilled water to remove unreacted hapten.
Although the number of diazo groups per hormone molecule could be
regulated by the number of moles of hapten and hormone reacted, a
parallel control experiment with S.sup.35 labelled sulfanilic acid
to evaluate the precise composition of the hapten-protein samples
was performed with each diazotization. The same hormone preparation
to be used for immunization was used in the control experiment.
After the reaction was completed, an aliquot was taken from the
reaction mixture and the remainder thoroughly dialyzed. Equal
volumes of the dialyzed and undialyzed solutions were counted by
liquid scintillation. By comparing the counts of the dialyzed and
undialyzed samples, the moles of hapten coupled to each mole of
hormone was calculated since the unreacted hapten was removed by
dialysis. For this calculation, a molecular weight of 30,000 was
assumed for all gonadotropin preparations.
Following dialysis, hapten-hormones were lyophilized and stored at
4.degree. C. Diazo-HCG (35 groups/molecule) and HLH (26
groups/molecule) were bioassayed by the ovarian ascorbic acid
depletion method and found to retain 62 and 85 percent respectively
of the activity of the unaltered hormones from which they were
derived. None of the other hormones were assayed for biological
activity.
Immunization Procedures
Female baboons received their initial immunization on days 3-5 of
the menstrual cycle and the second and third injections one week
apart. The fourth injection was given 2-3 weeks after the third. A
few animals received a fifth injection at 70-80 days after the
first injections. All antigens were administered subcutaneously in
a suspension of mannide manoleate or peanut oil. Doses of antigens
for each injection varied between 3 and 5 mg. Injection sites were
inspected daily for 5 days after each immunization for local
reactions.
Monitoring Effects of Immunization.
Daily 24-hour urine specimens and frequent serum samples were
collected during at least one menstrual cycle prior to
immunizations and following immunizations until the effects of
treatment were assessed. Urinary LH, urinary estrogens and plasma
progestins were measured. Antibodies were detected in
post-immunization serum samples by reacting 0.2 ml. of a 1:1000
dilution of serum in phosphate-buffered saline (pH 7.4) 0.5 percent
normal baboon serum with 250 pg of 1.sup.131 labelled hormone. Sera
were reacted with both the unaltered immunizing hormone and
unaltered baboon LH for antibody detection. A purified baboon LH
preparation (1.9.times.NIH-LH-S1) was used as a tracer antigen.
Antigen-antibody complexes were precipitated with ovine anti-baboon
gamma globulin after a 24-hr. incubation at 4.degree. C. Antibody
levels were expressed as pg of labelled hormone bound. Significant
antibody levels were considered to be those that would bind 5.0 pg
or more of the 1.sup.131 labelled antigen.
Antisera were fractionated by gel filtration of Sephadex G-200
according to the procedure of Fahey and Terry (at p. 36,
Experimental Immunology, F. A. Davis Co., Philadelphia, Pa., 1967,
incorporated by reference to the extent necessary to understand the
invention) to determine the proportion of IgM and IgG antibodies in
the baboon sera. Since the IgG fraction in this procedure contained
a portion of IgA and IgD antibodies, only IgM and total titers were
determined. The IgM fraction from the column was reacted with
1.sup.131 hormones and the binding capacity determined. The volumes
of the fractionated sera were adjusted so that antibody levels
would be comparable to those of whole serum.
Antibody Production
No significant reactions were observed at the site of injection
following any immunization. On 4 occasions, a slight induration
(2-3 cm in diameter) was seen when mannide manoleate was used as a
vehicle but the redness and swelling disappeared within 4-5 days.
Antibodies were detected against the immunizing antigen within 3-5
weeks in all animals. The extent, duration and cross reactivity of
these antibodies is recorded. Generally speaking, higher levels
were observed to heterologous gonadotropin immunization than to
homologous ones.
The cross-reactivity of induced antibodies with baboon LH was
studied on each animal. Cross-reactivity of antisera at peak levels
was recorded. Although relatively high antibody activity against
human LH and HCG were seen, relatively little reaction with baboon
LH occurred. An intermediate cross-reaction was noted with
anti-ovine LH and a high degree of cross-reactivity was seen with
anti-monkey LH. Diazo-human FSH was weakly antigenic in the baboon.
The duration of antibody production was generally longer with the
human and sheep gonadotropin immunization than with those of monkey
or baboon origin.
Peak antibody levels usually occurred at the time when the
antibodies had shifted to principally the IgG type. Early
antibodies had a larger proportion of IgM type and were generally
more cross-reactive with baboon LH. The change in the proportion of
the total antibody population that was IgM was recorded from the
time antibodies were first detected. Significant cross-reactivity
to baboon LH was observed in anti-human gonadotropins when IgM was
abundant but dropped sharply as the antisera shifted to nearly all
IgG. This drop in cross-reactivity did not occur with monkey and
baboon immunizations. Again, the ovine LH immunization produced an
intermediate change in reactivity with the shift from IgM to
IgG.
Effects on the Menstrual Cycle
The effects of immunization upon the event of the menstrual cycle
were determined by observing changes in sex skin turgescence and
levels of pituitary and/or ovarian hormones. Based on these
parameters, the delay or retardation of ovulation from the expected
time, as judged by the control cycle, was calculated. One animal
immunized with HCG had no interruption in ovulation and another
immunized with HFSH was delayed for only one cycle. Two animals
injected with HLH and two injected with HCG had ovulation delays
equivalent to two menstrual cycles. A third animal immunized with
HLH was delayed a calculated 86 days. Ovine LH immunizations
produced an 88 day delay in ovulation.
Immunizations with diazo-monkey or baboon LH resulted in longer
disruption of the menstrual cycle. Calculated delays in ovulation
for the two animals receiving monkey LH was 146 and 122 days
whereas the animals receiving altered baboon LH were retarded from
ovulation 224 and 210 days.
Effects on specific hormone patterns following immunization with
HLH in one animal were recorded. The interval between menses was
considered to represent a "cycle". Urinary estrogens and plasma
progestin patterns indicated that no ovulation occurred during the
cycle of immunization which was 85 days in duration. Urinary
estrogens were elevated during treatment but did not reflect a
typical pattern. Plasma progestins were not elevated until about
day 19 of the first post-treatment cycle. Patterns of both
estrogens and progestins were within normal limits during the
second post-treatment cycle. Antibody levels were elevated from
about day 35 of the treatment cycle until 289 days from the first
detection of antibodies. An LH assay was not available when this
animal was studied and no data on plasma or urinary levels of this
hormone was obtained.
Hormonal patterns following an immunization with diazobaboon LH
were recorded. In this animal, antibody levels were lower and
persisted, in general, for a shorter period than did immunizations
with human gonadotropins. During the treatment cycle, levels of
urinary estrogens and plasma progestins followed a normal pattern
but were quantitatively lower than normal. Urinary LH patterns
fluctuated markedly due to the injections of diazo-LH during this
period. No conclusive evidence of ovulation was obtained for the
treatment cycle. The first post-treatment cycle lasted 246 days.
During this cycle urinary LH and estrogens were elevated on days
35-41 but there was no subsequent elevation in plasma progestins
that would indicate ovulation had occurred. Following day 42 of
this cycle, there was no significant elevation in any of the three
hormone levels until day 231 when significant elevations of urinary
estrogens and LH occurred. These rises were followed 3 days later
by an elevation in plasma progestins indicating the presence of a
functioning corpus luteum. A second post-treatment menstrual cycle
was of normal duration and the endocrine patterns were normal.
Antibodies to unaltered baboon LH attained maximum levels by about
day 70 of the post-treatment cycle and remained relatively constant
until day 190 when a steady decline was observed. By day 215 of
this cycle, antibody levels were barely detectable. Approximately
16 days after this time, a peak of LH commensurate with a normal
midcycle elevation was observed. From this point the animal
appeared to have the normal function of the pituitary-ovarian axis.
Hormonal patterns in animals with other heterologous gonadotropin
immunizations were similar to animal receiving HLH and other
animals receiving monkey or baboon LH were similar in response to
animal receiving baboon LH.
These results in baboons indicated that the modification of a
reproductive hormone, by the procedures outlined, did render it
antigenic and the antibodies thus formed did neutralize natural
endogenous hormones if the natural hormone was obtained from the
species receiving the immunizations with modified hormone.
EXAMPLE II
HCG is a hormone naturally present only in pregnant women with the
exception that an entity at least analogous thereto has been found
to be present in humans in conjunction with neoplasms. HCG is also
commercially available. Human LH is immunologically and
biologically identical to HCG, even though there are chemical
differences. Since they are biologically identical and HCG is
readily available from commercial sources it was presumed that the
effectiveness of this immunological procedure could be evaluated by
injecting modified HCG into non-pregnant women and monitoring the
blood levels of LH. Antibodies formed will neutralize both the LH
and the modified HCG. Reference in the above regard is made to the
publications identified earlier herein.
Women have a pattern of LH levels; the level is substantially
constant until the middle period between menstrual cycles,
immediately prior to ovulation; at that point of LH level rises
greatly and helps induce the ovulation. Monitoring the LH level and
the antibody level will show that the procedure used did or did not
cause the production of antibodies capable of neutralizing the
endogenous reproductive hormone, namely LH.
A woman aged 27 years was selected for study. Hormone was obtained,
purified and modified. The modified human hormone (HCG) was
injected into the subject. It is well known that antibodies to HCG
react identically to LH as well as HCG. The effect of the
immunization was evaluated, principally by monitoring blood levels
of LH. Finally the results were evaluated.
Preparation of Hormone
Clinical grade HCG derived from pregnancy urine was obtained from
the Vitamerican Corp., Little Falls, N.J. This material has an
immunological potency of 2600 IU/mg. Contaminants were detected in
this preparation. Purification consisted of chromatography and
elution. Fractions were dialyzed and lyophylized. The most potent
fraction contained approximately 7600 IU/mg., however, it was
heterogenous on poloyacrylamide gel electrophoresis.
The fraction was further purified by gel filtration. The elution
profile revealed two major protein peaks. The most potent HCG was
found in the first peak and had an immunological potency of 13,670
IU per mg. This fraction was subjected to polyacrylamide gel
electrophoresis. Further purification by gel filtration showed no
evidence of heterogeneity of the HCG at this stage. Consequently,
materials for study were processed according to the above
procedure.
The contamination of this purified HCG was tested with I.sup.131
used for identification and a sample was reacted with antisera
against several proteins offering potential contamination. Those
proteins were follicle stimulating hormone, human growth hormone,
whole human serum, human albumin, transferin, alpha one globulin,
alpha two globulin and orosomucoid. No detectable binding of the
purified HCG was observed with any antisera at a dulution of 1:50
of each. These negative results, calculated against potential
binding of the respective proteins, indicated that contamination
with any was less than 0.005 percent.
Alteration of Hormone
Hormone was altered by coupling with a hapten (sulfanilazo). This
method couples the hapten molecules to the protein via the amino
group of the liphatic or aromatic portion of the hapten. The number
of hapten molecules coupled to each HCG molecule (Ha-HCG) can be
regulated and for this study, forty haptenic groups per HCG
molecule were used for preparing the immunizing antigen.
Following the hapten-coupling process, the Ha-HCG was sterilized
and tested.
Subject
The subject was multiparous and had terminated her reproductive
capabilities by prior elective bilateral salpingectomy. She was in
good health and had regular cyclic menstruation. She underwent
complete history, physical exmination and laboratory evaluation
including blood count, urinalysis, latex fixation and Papanicolau
smear. She had no history of allergy.
To demonstrate normal functioning of the pituitary-ovarian axis
prior to immunization, blood samples were obtained every other day
from the first day of menses for 10 days, then daily for 10 days
and finally, every other day until the next menses. Serum
determination of FSH, LH, estrone, estradiol and progesterone were
performed. These studies indicated an ovulatory pattern.
Immunization Procedures
Ten mg. of the Ha-HCG antigen were dissolved in 1.0 ml. of saline
and emulsified with an equal volume of oil. Prior to injection,
scratch tests to antigen and vehicle were performed. Immunizations
were begun in the luteal phase of the treatment cycle to prevent
superovulation from the administered HCG. Four injections at two
week intervals were given to the subject. The first two of these
were administered in oil subcutaneously (1.0 ml. in each upper
arm); the final two injections were given in saline only via the
intradermal route. Following each injection, blood pressure
readings were taken and the subject observed for allergic
reactions.
Monitoring Effects of Immunizations
Blood samples were collected at weekly intervals beginning two
weeks after the initial injection to test for the presence of
humoral and cellular antibodies. Following completion of the
immunization schedule, blood samples were collected in the same
manner as in the control cycle to assess effects of immunization on
hormonal patterns of the menstrual cycle. Since antibodies to HCG
react identically to LH as with HCG, LH was monitored as an index
of effectiveness of the procedure. A third cycle was similarly
studied six months after initial immunization. Upon completion of
the study, physical and pelvic examinations and laboratory
evaluations were repeated.
Serum samples from the control and post-treatment cycles were
assayed for FSH, LH, estrone, estradiol and progesterone.
The subject was tested for delayed hypertensivity before
immunization and at two week intervals until the injection schedule
was completed by an in vitro lymphocyte transformation test.
Results
Temporal relationships of serum pituitary and gonadal hormones in
the control cycles of the subject were recorded. Antibody titers to
HCG were detected in the subject after two injections. Menses
occurred at regular intervals during the immunizations.
Following the initial injection in mannide manoleate, some itching
and swelling at the injection site occurred. Subsequent intradermal
injections in saline produced no reactions and it was concluded
that the local reactions were induced by the mannide manoleate.
Lymphocyte transformation tests on plasma samples were
negative.
In the post-treatment cycle, baseline follicular and luteal phase
LH levels were not noticeably changed in the subject. Very small
midcycle elevation in LH levels were observed as compared to the
normal large increases. FSH patterns in the post-treatment cycle
were normal. This indicated that the antibodies were neutralizing
the action of endogenous LH.
The subject showed an ovulatory progesterone pattern but attained
relatively high antibody titers to LH and HCG after only two
injections of Ha-HCG.
The subject was studied during another cycle approximately six
months from the first immunization. Significant antibody titers
were found. LH patterns indicated a small midcycle elevation. FSH
patterns were essentially normal. Thus, the specificity of anti-HCG
antibodies to LH was shown but not to FSH.
EXAMPLE III
Another woman aged 29 years was selected for further study. Hormone
was obtained, purified, and modified as in Example II. This
modified hormone was injected into this subject in the same way as
in Example II. The subject was monitored and tested as in Example
II.
The results were similar to the results found in Example II except
that (1) the levels of estrone and estradiol were substantially
normal, (2) the subject acquired significant antibody titers late
in the post-immunization cycle, and (3) in the cycle studied after
six months this subject showed no significant midcycle elevation in
LH patters.
EXAMPLE IV
Another woman aged 29 years was selected for further study. Hormone
was obtained and purified and modified as in Example II. This
modified hormone was injected into this subject in the same way as
in Example II. The subject was monitored and tested as in Example
II.
The results were similar to the results found in Example II except
that (1) baseline follicular and luteal phase LH levels were
noticeably depressed in the post-treatment cycle, (2) no midcycle
elevations were observed in LH, (3) estrone levels were elevated
during the follicular phase of the post-immunization cycle, and (4)
during the six-months study there was no significant midcycle
elevation in LH patterns.
EXAMPLE V
Another woman aged 35 years was selected for further study. Hormone
was obtained, purified, and modified as in Example II. This
modified hormone was injected into this subject in the same way as
in Example II. The subject was monitored and tested as in Example
II.
The results were similar to the results found in Example II except
that (1) baseline follicular and luteal phase LH levels were
noticeably depressed in the post-treatment cycle, (2) a very small
midcycle elevation of LH was observed, (3) levels of FSH patterns
in the post-treatment cycle were depressed, and (4) levels of both
estrone and estradiol were reduced, during the follicular phase of
the post-immunization.
EXAMPLE VI
Another woman aged 28 years was selected for further study. Hormone
was obtained, purified, and modified as in Example II. This
modified hormone was injected into this subject in the same way as
in Example II. The subject was monitored and tested as in Example
II.
The results were similar to results found in Example II except that
(1) baseline follicular and luteal phase LH levels were depressed
in the post-treatment cycle, (2) no peaks were observed in midcycle
levels of LH, (3) estrone levels appeared elevated in the
follicular phase of the post immunization cycle, and (4) LH
patterns indicated no significant midcycle elevation in the
six-month post-immunization cycle.
EXAMPLE VII
Another woman aged 28 was selected for further study. Hormone was
obtained, purified, and modified as in Example II. This modified
hormone was injected into this subject in the same way as in
Example II. The subject was monitored and tested as in Example
II.
The results were similar to results found in Example II except that
(1) antibody titers to HCG were not detected until after three
injections, (2) baseline follicular and luteal phase LH levels were
depressed in the post-treatment cycle, (3) no peaks nor midcycle
elevation in the LH were observed, (4) estrone levels were elevated
during the follicular phase, and (5) no significant antibody titers
were found in the six-month cycle.
All the above examples show the practicality of injecting modified
hormones for the purpose of neutralizing an endogenous reproductive
hormone and thereby offering a procedure for the prevention of
conception or the disruption of gestation.
EXAMPLE VIII
Data obtained in earlier experiments and discussed in Examples
I-VII showed that a modified natural reproductive hormone, when
injected into an animal of species from which it was derived, would
produce antibodies that would neutralize the action of the
unmodified endogenous natural hormone in the body of the animal.
Hormones used in Examples I-VII were FSH, LH and HCG. New
experiments were performed, based on this knowledge, to identify
another reproductive hormone (placental lactogen) that could be
used in a similar fashion.
Preparation of Hormone
A purified preparation of placental lactogen was prepared from
placentae of baboons since it was intended to use modified
placental lactogen to immunize baboons. Placentae were extracted
and purified on column chromatograph according to previously
published procedures. The purity was tested by polyacrylamide gel,
electrophoresis and by radioimmunoassay. The material obtained
showed a high degree of purity on electrophoresis and
radioimmunoassay showed no contamination with other placental
hormones.
Hormone Modification and Immunizations
The baboon placental lactogen (BPL) was altered by coupling with
the diazonium salt of sulfanilic acid as outlined for other
hormones in Example I. The number of diazo molecules per BPL
molecule in this instance was 15. Immunization procedures were also
similar to those described in Example I for other hormones.
Results
Within 4-6 weeks after the first injection of diazo-BPL, antibody
levels to natural unmodified BPL in vitro were detected in 6 female
baboons. Levels rose to a plateau within 8-10 weeks and remained
there for several months. Hormonal measurements indicated that
there were no effects on the normal events of the menstrual cycle
due to the immunizations. Since BPL is normally secreted only in
pregnancy, this was not a surprising observation.
All six females were mated with a male of proven fertility three
times (once each in three different cycles during the fertile
period). Pregnancy diagnosis by hormonal measurement was performed
after each mating. From the 18 matings, there were 13 conceptions
as judged by pregnancy tests. The animals that were pregnant had
menstrual bleeding 7-12 days later than was expected for their
normal menstrual cycles. Subsequent hormonal measurements confirmed
that these 13 pregnancies were terminated by abortions
approximately one week after the time of expected menses.
These findings suggest that the antibodies formed in the animals
body after immunization had no effect on the nonpregnant menstrual
cycle but when pregnancy was established, they neutralized the
baboon placental lactogen in the baboon placenta and the result was
abortion very early after conception.
When in Examples I-VIII above Structures (I), (II), (III) are
modified by use of diazosulfanilic acid, dinitrophenol, or S-aceto
mercaptosuccinic anhydride or Structures (II), (III) are modified
by addition of polytyrosine or polyalanine, according to known
methods, the results obtained shall be similar to those in said
Examples.
Similarly, when FSH, somatomedian, growth hormone or angiotension
II are modified by use of diazosulfanilic acid or trinitrophenol,
the results obtainable upon administration of the purified modified
polypeptide into a male or female human or animal would indicate
the stimulation of antibodies which neutralizes all or some of the
modified polypeptide as well as corresponding endogenous
polypeptide.
EXAMPLE IX
The subjects used in the studies reported in the example are female
baboons. All baboons were adults of reproductive age. A description
of subjects and the conditions of experimentation have been
described in Example I. The animals have been studied using highly
purified beta subunits of HCG using a preparation with a biological
activity of less than 1.0 IU/mg. Animals were immunized with 14-26
moles/mole of polypeptide of diazosulfanilic acid coupled subunits
in mannide manoleate.
Antibody levels were assessed by determining the binding of serum
dilutions with I.sup.125 labelled antigens. Cross-reactivity of
antisera was measured by direct binding of labelled antigens and by
displacement radioimmunoassays. Antifertility effects in actively
immunized animals were tested by mating females with males of
proven fertility. Effects in pregnant baboons passively immunized
with either sheep or baboon anti-.beta.-HCG were determined by
monitoring serum levels of gonadotropins and sex steroid hormones
before and after immunizations.
Eight female baboons were immunized with the modified beta subunit
of HCG. Significant antibody levels were attained in all
animals.
Baboon immunizations with the modified beta subunit of HCG resulted
in high antibody levels reacting to HCG, human LH and baboon CG but
not to baboon LH. All animals remained ovulatory, however, no
pregnancies resulted from numerous matings with males of proven
fertility. Passive immunization of non-immunized pregnant baboons
with sheep anti-.beta.-HCG serum produced abortions within 36-44
hours.
EXAMPLE X
Hemocyanin from Keyhole limpet (KLH) solution (7 mg/ml) in 0.05 M
sodium phosphate buffer in 0.2 M NaCl, pH 7.5, is prepared.
Insoluble particles are removed by centrifugation. To one ml of
this solution, tolylene diisocyanate (T.D.I.C.) reagent is added
(20 .mu.l) diluted to 1/30 with dioxane, the amount being
essentially the equivalent of the moles of lysyl residues in the
KLH molecules. After 40 minutes at 0.degree. C., the T.D.I.C.
activated KLH solution is combined with 0.5 mg of synthetic
.beta.-HCG peptide having the following structure:
Asp-His-Pro-Leu-Thr-Cys-Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Ser-Ser-Ser-Lys-Als
-Pro-Pro-Pro-Ser-Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pro-Gly-Pro-Pro-Asp-Thr-Pro-Il
e-Leu-Pro-Gln-Ser-Leu-Pro
Structure (XV)
which is first dissolved in 25 .mu.l of 0.05 M sodium phosphate
buffer in 0.2 M, NaCL, pH 7.5. The mixture is incubated at
37.degree. C. for four hours. The resulting product is purified by
gel filtration.
EXAMPLE XI
One g. of Ficoll 70 is dissolved in 1 ml each of normal saline and
2 M ethylene diamine (adjusted to pH 10 with hydrochloric acid)
solution. The solution is kept at room temperature in a water bath
and stirred with a magnetic stirrer. Cyanogen bromide, 4 g,
dissolved in 8 ml of dioxane, is added to the Ficoll 70 solution.
The acidity of the mixture is maintained at pH 10-10.5 for 8
minutes by adding drops of 2 N sodium hydroxide solution. An
additional 2 ml of 2 M ethylene diamine, pH 10, solution is added,
and stirring at room temperature is continued for 30 more minutes.
The product is purified by passing it through a Bio-Gel p-60
column.
EXAMPLE XII
Two mg of the compound of Structure (II) containing picogram amount
of I.sup.125 labeled adduct and KLH (1.6 mg) of dissolved in 1 ml.
of 1.0 M glycine methyl ester in 5 M guanidine hydrochloride. Ethyl
dimethylamino propylcarbodiimide (E.D.C.) 19.1 mg is added to this
solution. The acidity is adjusted to and maintained at pH 4.75 with
1 N HCl at room temperature for 5 hours. The KLH-peptide conjugate
is purified by passing it through a Bio-Gel p-60 2.2.times.28 cm
column equilibrated with 0.2 M NaCl.
EXAMPLE XIII
Solid bifunctional imidoester dihydrochloride (3 mole) is added in
2 mg portions at 5-minute intervals to a constantly stirred
solution of 1 mole of polypeptide of Structure (II) (1-20 mg/ml) in
0.1 M sodium phosphate, pH 10.5, at room temperature. Sodium
hydroxide 0.1 N is added to maintain the acidity at pH 10.5. One
hour after the addition of the diimidoester has been completed, a
polymerized product according to this invention is obtained.
EXAMPLE XIV
To a 20 mg/ml solution of homologous serum albumin in 0.1 M borate
buffer, pH 8.5, 1000% mole excess of 25% aqueous solution of
glutaric dialdehyde is added at room temperature. The excess
dialdehyde is removed by gel filtration in water using Bio-Gel p-2.
The material collected at the void volume is lyophilized, and the
dried product is redissolved in 0.1 M borate buffer, pH 8.5 (20
mg/ml), mixed with the required amount of polypeptide of the
following Structure:
Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Ser-Ser-Ser-Lys-Als-Pro-Pro-Pro-Ser-Leu-Pro
-Ser-Pro-Ser-Arg-Leu-Pro-Gly-Pro-Pro-Asp-Thr-Pro-Ile-Leu-Pro-Gln-Ser-Leu-Pr
o
Structure (XVI)
(20 mg/ml) in the same buffer at room temperature. Twenty minutes
later, sodium borohydride in 250 percent molar excess of
polypeptide XVI is added. The reaction is terminated after one
hour. The conjugated product is purified by gel filtration on
Bio-Gel p-60 column, dialyzed free of salt and lyophilized.
EXAMPLE XV
Ficoll 70 1 g, NaHCO.sub.3 500 mg, cyanuric chloride 3 g, H.sub.2 O
20 ml, and dimethylformamide 80 ml., are stirred at temperature
below 16.degree. C. for 2 hours. The product is dialyzed against
distilled water until Cl-free, then lyophilized. A polypeptide of
Structure (XV) (2 mg) containing minute quantity of I.sup.125
-labeled analogue is incubated with 1 mg of this product in 0.25 ml
of 0.2 M sodium borate buffer, pH 9.5, for one hour at 20.degree.
C., and the product is recovered from a Bio-Gel p-60 2.2.times.28
cm column.
When the above procedure is carried out and Dextran T 70 is used in
place of Ficoll 70, the corresponding modified polypeptide, useful
according to this disclosure, is obtained.
EXAMPLE XVI
Ficoll 70 1 g, NaIO.sub.4 1.2 g, and KCl 0.42 g are dissolved in
1.5 ml of 1 M sodium acetate buffer, pH 4.5, and incubated at
37.degree. C. for 1 hour.
Two mg (=588 .mu.moles) of polypeptide of Structure (XV) above
mixed with minute quantity of I.sup.125 -labeled analogue is
incubated with 2 mg of the product obtained above in 0.3 ml of 0.2
M borate buffer, pH 9.5 at 55.degree. C. for 1 hour. The reaction
mixture is then chilled in an ice water bath and NaBH.sub.4 1 mg is
then added into this solution. The reduction reaction is terminated
by passing the product through a Bio-Gel p-60 2.2.times.28 cm
column equilibrated and eluted with 0.2 M NaCl.
EXAMPLE XVII
Numerous rabbits are immunized with a variety of synthetic peptides
conjugated to different modifying groups. Following two or three
immunizations at 3-5 week intervals, sera from animals are assessed
by determining their ability to bind in vitro to radiolabeled HCG.
The specificity of this binding is studied by reacting the same
sera against similarly labeled other protein hormones,
particularly, pituitary LH. Sera are further assessed by
determining their ability to inhibit the biological action of
exogenously administered HCG in bioassay animals. Thus, the
increase in uterine weight of the immature female rat in response
to a prescribed dose of HCG is noted. The dose of HCG is
administered subcutaneously in saline in five injections over a
three day period and the animal is sacrificed for removal of the
uterus on the fourth day. The weight of the uterus increases in
dose reponse fashion to the hormone injections. When assessing the
effects of antisera in this response, varying quantities of test
serum are administered intraperitoneally separately from the
subcutaneous injection of hormone during the assay. This procedure
permits the antiserum to be absorbed rapidly into the rat's
bloodstream and will permit interaction of it with hormone when the
latter likewise enters this fluid. If the antiserum is capable of
reacting with the hormone in a manner preventing stimulation of the
uterus, the antiserum is considered to be effective for biological
inhibition of hormone action.
The frequency of animals showing a positive response to
immunological binding and neutralization of biological activity is
presented in
EXAMPLE XVIII
Iodosobenzoic acid dissolved in a slight excess of 1 N potassium
hydroxide in 10% molar excess is added to the peptide of Structure
(II) in phosphate buffer with normal saline at pH of 7.0. After
thirty minutes at room temperature, the product polypeptide dimer
is purified by gel filtration.
EXAMPLE XIX
To an ice water bath cooled and vigorously stirred 0.23 ml. of
bovine gamma globulin (10 mg/ml) in 0.05 M phosphate buffer with
normal saline (PBS) pH 7.5, 50 .mu.l of 1/10 T.D.I.C. in dioxane is
added. After 40 minutes, the excess T.D.I.C. is removed by
centrifugation (0.degree. C., 10 minutes, 10,000 g) and the
precipitate is washed twice with 0.1 ml. of PBS. The combined
supernatents are added to 7.7 mg. of the peptide of structure II
dissolved in 0.8 ml. of PBS, pH 7.5. The mixture is stirred at room
temperature for 10 minutes, then incubated at 37.degree. C. for 4
hours. The conjugate product is purified by dialysis.
EXAMPLE XX
BSA (10 mg/ml) in PBS solution (0.25 ml.) is treated with 50 .mu.l
of 1/10 T.D.I.C. dioxane solution and conjugated to 7.5 mg. of
synthetic .beta.-HCG peptide of Structure (III) in 0.8 ml. of PBS
(pH 7.5) as in Example XIX to obtain the product.
EXAMPLE XXI
To an ice water bath cooled and vigorously stirred 0.6 ml. of
.beta.-HCG peptide of Structure (III) (10 mg/ml) in phosphate
buffered saline, pH 7.5, is added 30 .mu.l of 1/10 T.D.I.C. After
40 minutes, the excess T.D.I.C. is removed by centrifugation
(10,000 g, 0.degree. C., 10 minutes) and the precipitate is washed
twice with 0.1 ml. PBS. The combined supernatents are added to 3
mg. of poly (D, L-Lys-Als) dissolved in 0.3 ml. of PBS. The mixture
is incubated at 37.degree. C. for 4 hours. The product is then
dialyzed and lyophilized.
EXAMPLE XXII
The results set out in Table I provide further evidence of the
broad applicability of this invention as indicated previously in
this specification.
Using standard methods of testing in rabbits, both immunological
binding response and neutralization of biological activity were
established for the modified polypeptides indicated with the result
as set out in Table I.
EXAMPLE XXIII
Antigen was prepared by reacting a Diisocyanate (T.D.I.C.--see
above) coupling reagent with carrier (tetanus toxoid), extracting
excess reagent and incubating activated carrier with peptide
Structure (II). Baboons were immunized with the antigen and the
results of mating 4 animals three times are shown in FIG. 1. The
figure shows that from 12 exposures (matings) one pregnancy
resulted even though relatively low levels of immunity from the
antigen were achieved. Non-immunized baboons of the same colony had
a fertility rate of approximately 85%.
EXAMPLE XXIV
Referring to FIG. 2, baboons were immunized initially with a beta
subunit of HCG modified by diazotization in a manner similar to
that described in conjunction with Example II. Following this
initial administration, the baboons were injected 21 and 42 days
later with Structure (II) above having been modified by the same
diazotization process. FIG. 2 shows plots representing the levels
of antibodies generated in consequence of these administrations.
Such quantities of antibodies are expressed as micrograms of
isotopically--labeled HCG that will bind each milliliter of serum
from the baboons at specified days after the initial injection. The
levels shown were maintained for a period of over one year.
TABLE 1
__________________________________________________________________________
Frequency of Positive Antibody Responses to Various HCG
Peptide-Conjugates Number of Rabbits Immunological Neutralization
of Peptide Carrier Immunized Binding Responses Biological Activity
__________________________________________________________________________
35 amino acid 111-145 Bovine Gamma Globulin 10 10 6 Morgan et al
Keyhole Limpet Peptide II Hemocyanin 10 5 * 31 amino acid 115-145
Poly-D-L-Alanine 10 9 5 Morgan et al Bovine Serum Albumin 12 12 6
Peptide III 44 amino acid 105-148 Keyhole Limpet Hemocyanin 10 8 *
Peptide XV Natural 109-145 Keyhole Limpet Keutman Hemocyanin 10 10
* Peptide XII
__________________________________________________________________________
*additional time needed for assessment
Referring to Table 2, the results of breeding the two baboons
represented in FIG. 2 is revealed in tabular form. The table
presents the results of mating these animals ten times over a
period of approximately one year. These data suggest that the
animals ovulated in every cycle, however, no pregnancy was
observed, as indicated by the animal having a menstrual period at
or before the expected time therefor. While the results tabulated
demonstrate the efficacy of the entire procedure, it was observed
for the particular structure utilized in the primary immunization,
i.e. Structure (I), antibody cross reactivity with LH was observed.
Such cross reactivity may be avoided by the utilization of the
fragment conjugation procedures set forth in detail
hereinabove.
EXAMPLE XXV
The specificity of antibody response to a CG
fragment-macromolecular carrier is represented by the instant
experiment. A 35 amino acid sequence [Structure (II), herein
"synthetic peptide"] of the HCG beta subunit was conjugated with
bovine gamma-globulin and administered to a baboon. Varying doses
of each of these three hormones were tested for their ability to
compete with I.sup.125 -labeled synthetic peptide [structure (II)]
bound to the antiserum. The results are set forth in FIG. 3. Note
from the figure that Human LH was ineffective for displacement of
tracer antigen at doses up to 2.5 IU (international units). Since
HCG displaced antigen at a dose of 20 mIU, the cross-reactivity
with HLH in this assay system was less than 0.8%. Baboon CG also
displaced I.sup.125 -labeled antigen in this assay and, based on
biological potency of the two hormones, was about 20% as effective
as HCG.
Table 2 ______________________________________ Breeding of
Immunized Baboons [Diazo-.beta.-HCG presensitized] Booster:
Diazo-.beta.-HCG-(111-145) 1 2 Pre-Mate Pre-Mate Titer Ovul. Preg.
Titer Ovul. Preg. ______________________________________ Mating No.
1 5.00 + - 4.20 + - Mating No. 2 4.25 + - 4.10 + - Mating No. 3
4.22 + - 4.00 + - Mating No. 4 4.17 + - 3.89 + - Mating No. 5 3.80
+ - 3.76 + - Mating No. 6 6.65 + - 5.00 + - Mating No. 7 5.90 + -
4.75 + - Mating No. 8 5.10 + - 4.20 + - Mating No. 9 5.00 + - 4.25
+ - Mating No. 10 4.66 + - 4.00 + -
______________________________________
EXAMPLE XXVI
The following experiments were carried out to determine whether the
carbohydrate chains contained in the C-terminal 37 residues of
.beta.-HCG influence the immunogenicity of that peptide.
A peptide representing amino acid residues 109-145 of .beta.-HCG
was isolated from a chymotriptic digest of reduced and
carboxymethylated .beta.-HCG by procedures reported by Keutmann, H.
T.; Williams, R. M., J. Biol. Chem. 252, 5393-5397 (1977). This
peptide is identified in Table 3 as P-1. The purity of the peptide
was confirmed by amino acid and terminal end group analyses. A
portion of the isolated peptide was treated with anhydrous
hydrofluoric acid (HF) to remove carbohydrate moieties and
repurified by column chromatography according to methods described
by Sakakibara S. et al, Bull. Chem. Soc. Japan, 40, 2164-2167
(1967). This portion of the isolated peptide is identified in Table
3 as P-2. Complete removal of the sugar chains were confirmed by
carbohydrate analysis; See Nelson, Norton, J. Biol. Chem. 153,
375-380 (1944). A third peptide with the amino acid sequence
109-145 of .beta.-HCG was prepared synthetically using the solid
state synthesis procedure of Tregear, G. W. et al., Biochem. 16,
2817 (1977). This third peptide is identified in Table 3 as P-3.
Highly purified HCG was used in all immunological experiments where
reference was made to intact HCG.
Preparation of Immunogens and Immunizations
Conjugates of the three peptides were prepared to keyhole-limpet
hemocyanin (KLH) using tolulene diisocyanate. A peptide-carrier
ratio of 4-6 peptides per 100,000 daltons of carrier was obtained
for different conjugates prepared according to amino acid analyses.
Rabbits were immunized with conjugates by three multiple site
intramuscular injections of 1.0 mg. of conjugate in 0.5 ml. of
saline emulsified with an equal volume of Freund's complete
adjuvant. Injections were given at 3 week intervals and weekly
blood samples were collected from 3-20 weeks of immunization.
Evaluation of Antisera
Antisera to all conjugates were monitored for antibody levels by
reacting dilutions of sera with I.sup.125 labeled HCG (chloramine T
method) at 4.degree. C. for 5 days and precipitating immune
complexes with sheep anti-rabbit gamma globulin serum. Antibody
levels were determined by assessing dilution curves in which a
linear correlation between dilution and binding of labelled antigen
at equilibrium occurred. At least 3 points in each curve were used
in calculating levels. These levels were expressed as .mu.g. HCG
bound per ml. of undiluted serum calculated by multiplying mass of
labelled antigen bound by serum dilution.
A radioimmunoassay system employing I.sup.125 HCG and antisera
raised to peptide conjugates was used to determine the relative
ability of HCG and peptides to compete with labeled HCG. Peak
antibody levels from each rabbit were evaluated in these studies.
Antigens and antisera contained in phosphate-buffered saline (pH
7.4) BSA (1%) were added to test tubes and incubated at 4.degree.
for 5 days. Separation of free and bound tracer HCG was
accomplished by the addition of sheep anti-rabbit gamma globulin
serum and further incubated for 48 hours followed by
centrifugation. Assessment of parallelism of dose response curves
was accomplished using methods described in Rodbard, D. in: Odell,
W. D. and Daughaday, W. H., eds., "Competitive Protein Binding
Assays," J. B. Lippincott, Phila. Pa. (1971). The ability of
unlabelled HCG and peptides to compete with I.sup.125 HCG for
antibody binding sites was expressed as moles of unlabeled antigen,
per mole of unlabeled HCG, required to reduce the binding of
labeled HCG by 50%. For this purpose molecular weights for HCG,
P-1, P-2, and P-3 of 38,000, 7,000, 3,990, and 3,990 respectively
were used. The molecular weight of the P-1 peptide was an estimate
since the contribution of the 4 carbohydrate chains to its size was
not determined. Four radioimmunoassays were performed with each of
the 11 antisera studied and the results presented as the mean of
the four values.
RESULTS
Parallel dose response curves of HCG and peptides were observed in
all radioimmunoassays. In the assay system employed, 200-400 moles
of unlabeled HCG was required per mole of labeled HCG at 50%
binding of the latter to antisera. There was no detectable
difference among antisera to the 3 peptide conjugates in the
ability of intact HCG to compete with labeled hormone for antibody
binding sites.
Data obtained from comparing the ability of HCG and peptides to
compete with I.sup.125 HCG for binding to anti-peptide sera
revealed some qualitative differences in the antisera (Table 2).
Much larger quantities of P-2 peptide and P-3 peptide were required
to reduce I.sup.125 HCG binding than was required by P-1 peptide
when sera against the P-1 peptide was tested. While similar
quantities of P-2 and P-3 peptides were required to inhibit one
mole of labeled HCG binding, these were 2-10 times the amounts
required by the P-1 peptide.
Differences in the quantities of peptides required to compete with
an equivalent mass of labeled HCG were less using antisera raised
to carbohydrate-free natural peptide (P-2). More P-1 peptide was
needed for an equal reduction in binding than the other 2 peptides.
No significant difference could be detected in the quantities of
P-2 or P-3 peptides required among the 3 antisera tested.
Approximately 1.5-2.0 times as much P-1 peptide was required to
compete equally with I.sup.125 HCG for antibodies raised to the P-3
peptide but P-2 peptide reacted nearly as well as did the synthetic
peptide.
Table 3 ______________________________________ Mean Quantities of
HCG and 109-145 C-terminal .beta.-HCG Peptides Required to Compete
with I.sup.125 HCG at 50% Binding of Labelled Hormone Unlabelled
Antigens Antisera HCG P-1 P-2 P-3 mol/mol mol/mol mol/mol mol/mol
Rabbit HCG I.sup.125 HCG I.sup.125 HCG I.sup.125 HCG I.sup.125 No.
(X .+-. SE) (X .+-. SE) (X .+-. SE) (X .+-. SE)
______________________________________ Anti P-1 78 284 (12.6) 430
(11.8) 4565 (200.8) 3628 (154.1) 79 350 (13.5) 404 (18.5) 855
(33.4) 881 (42.2) 171 403 (17.7) 343 (9.9) 899 (35.1) 759 (37.1)
173 377 (16.5) 320 (13.9) 1448 (72.4) 1536 (73.7) Anti P-2 93 247
(11.8) 385 (18.2) 264 (12.5) 268 (12.73) 94 294 (14.1) 431 (15.5)
362 (15.2) 329 (13.8) 252 201 (9.6) 296 (12.4) 216 (7.7) 205 (9.0)
Anti P-3 405 496 (23.6) 998 (47.4) 628 (27.6) 309 (13.6) 411 489
(20.5) 1200 (50.4) 678 (29.7) 413 (16.1) 416 364 (13.1) 581 (20.9)
400 (14.4) 271 (12.8) 417 340 (14.9) 474 (18.4) 176 (6.8) 105 (4.6)
______________________________________
DISCUSSION
Despite low levels of antibodies obtained in this study, the
carbohydrate-containing peptide was not more immunogenic than those
without this moiety when conjugates to both were prepared in the
same manner.
From these studies, it can be concluded that although antibodies to
carbohydrate free peptides are qualitatively different than those
to the natural peptide, antisera generated to the synthetic peptide
reacted with HCG as well as antisera to natural peptides and
equivalent to natural and synthetic peptides elicited similar
anti-HCG levels in rabbits.
EXAMPLE XXVII
In this Example, a polypeptide fragment structure having an --SH
group is activated utilizing the following reagent: ##STR8## A
solution of the reagent (1.2 eq. per --SH group in the polypeptide)
in a suitable water miscible organic solvent, such as dioxane, is
added to a solution of the polypeptide fragment structure, e.g.
Structure (XII) (which has had its amino groups blocked) in aqueous
buffer at pH 6.5. After 2 hours, the solvent is removed at a
temperature of less than 30.degree. C. under vacuum, and to the
residue are added water and ethyl ether (1:1). The aqueous layer is
separated and its pH adjusted to approximately 8.5 by the addition
of sodium hydroxide solution and this alkaline mixture is added
rapidly to an aqueous solution of the carrier, e.g. the above
described influenza subunit, maintained at pH 8.5 by a suitable
buffer. After a further 4 hours, the conjugate is isolated, by gel
filtration.
EXAMPLE XXVIII
With the following reagent: ##STR9## a solution or suspension of a
carrier containing no sulfhydryl groups such as Flagellin in a
suitable aqueous buffer at a pH 6.5 is treated with the required
(1.2 eq/--NH.sub.2 desired to be reacted) amount of a solution of
the reagent in dimethylformamide. After 1 hour, the modified
carrier is isolated by column chromatography and added to buffer at
pH 6-7. This is then treated with a solution of the selected
fragment (containing sulfhydryl groups) in the same buffer and the
reaction is allowed to proceed for 12 hours before the conjugate is
isolated by column chromatography.
EXAMPLE XXIX
Modification of non-sulfhydryl containing peptide fragments [e.g.
structure (II)] or a carrier such as Flagellin to a sulfhydryl
containing one via "thiolactonization" is carried out as
follows.
The peptide is dissolved in a 1 M aqueous solution of imidazole
containing 0.5% of ethylenediamine tetraacetic acid at a pH of 9.3
under an atmosphere of nitrogen and a 100 fold excess of
N-acetylhomocysteine thiolactone is added in three portions at
eight hour intervals. After a total of 30 hours, the pH is adjusted
to 3-4 with acetic acid and the modified peptide is isolated by gel
chromatography and elution with 0.5 M acetic acid.
EXAMPLE XXX
The carrier protein is reacted with the N-hydroxysuccinimide ester
of a halo-(either chloro, bromo or iodo) acetic acid in the general
procedure described in the first part of Example XXVIII thus
yielding a modified carrier containing the required number of
halomethyl alkylating groups as desired.
To a solution of the sulfhydryl containing peptide [e.g. structure
(XII)] in a phosphate buffer at pH 6.5-7.0 under nitrogen at room
temperature is added an aqueous solution or suspension of the
modified carrier prepared above. The mixture is stirred for 12
hours. It is then washed with ethyl acetate and the conjugate
contained in the aqueous phase is purified by dialysis, gel
chromatography and lyophilization.
Should neither the carrier nor polypeptide fragment contain a
sulfhydryl group, one may be introduced into either of them by the
standard procedures such as "thiolactonization" described above
under Example XXIX.
* * * * *